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Protein phosphorylation maintains the normal function of cloned human Cav2.3 channels

Cav2.3 Ca2+ channels are subject to cytosolic regulation, which has been difficult to characterize in native cells. Neumaier et al. demonstrate the role of phosphorylation in the function of these channels and suggest a close relationship between voltage dependence and the phosphorylation state.

Introduction
Electrophysiological recordings from excised cell patches or dialyzed cells are almost invariably hampered by time-dependent changes in voltage-gated ion channel function. The most common form of these phenomena, termed run-down or washout, is a progressive decline of ionic currents and is thought to reflect changes in intracellular signaling cascades, which occur secondary to the loss or dilution of cytosolic factors (Becq, 1996). It can be preceded by a transient current facilitation (run-up), which may reflect voltage-and time-dependent repriming (i.e., recovery from inactivation) or modification of signaling cascades that tonically inhibit these currents (Tiaho et al., 1993;Elhamdani et al., 1994Elhamdani et al., , 1995. Although run-down remains a major obstacle for studies on voltage-gated Ca 2+ channel (VGCC) function, it has also provided insight into the manifold regulation of these channels in intact cells. For example, the decline of L-type Ca 2+ currents has been linked to several interrelated processes, which may include loss of ATP and other cytoplasmic factors, progressive protein dephosphorylation, decoupling of guanosine-5′-triphosphate (GTP)-binding proteins, and possibly increased proteolysis of the channels (Chad et al., 1987;McDonald et al., 1994;Kepplinger and Romanin, 2005;Xu et al., 2016;Yu et al., 2016). In P/Q-, N-, and certain neuronal L-type Ca 2+ channels on the other hand, run-down appears to involve depletion of membrane PIP 2 , a mechanism also thought to mediate M 1 muscarinic receptor-dependent inhibition of these channels (Wu et al., 2002;Suh et al., 2010). Much less is known about the run-down of "pharmaco-resistant" R-type currents, which are mainly mediated by Ca v 2.3-type VGCCs. R-type and R-type-like currents have been shown to exhibit both run-up and run-down (Cota, 1986;Hilaire et al., 1997;Benquet et al., 1999;Almog and Korngreen, 2009), but low expression levels and the need for pharmacological isolation have generally prevented further characterization of the two processes in native cells. The human embryonic kidney (HEK-293) cell line is widely used for heterologous expression of recombinant ion channels and receptors because it contains few endogenous channels, whereas most signaling pathways for regulation and posttranslational processing are operational (Toth et al., 1996;Thomas and Smart, 2005;Clare, 2006). Apart from circumventing the need for R-type current isolation, HEK-293 cells have a simple and uniform shape, which facilitates reproducible manipulation of their intracellular milieu. We therefore used conventional and perforated-patch-clamp recordings together with different inhibitors and cytosolic factors to study the effects of cell dialysis in a stably transfected HEK-293 cell line expressing human Ca v 2.3+β 3 channel subunits. Our findings show that the decline of macroscopic currents during run-down can partly be accounted for by changes in channel voltage dependence and that it can be prevented or slowed down by provision of intracellular ATP and in perforated-patch recordings. Protection from run-down depended on ATP-hydrolysis and was not related to lipid kinase-mediated PIP 2 synthesis or phosphorylation of tyrosine residues but was sensitive to inhibition of serine/threonine kinases. Protein kinase inhibition in intact cells also reduced peak current densities and reproduced the effects of run-down on channel voltage-dependence. Together, these findings indicate that run-down involves constitutive dephosphorylation of sites on the channels themselves or an associated protein and that ATP promotes phosphorylation of these sites by one or more endogenous kinases. Interestingly, our findings also indicate that the current facilitation during run-up could involve activation of leupeptin (Leu)-sensitive proteases, which may also influence the protective action of ATP.

Electrophysiological recordings
Cells were voltage-clamped by using the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981). Pipettes were prepared from thick-walled borosilicate glass capillaries (1.5/0.84-mm-outside/inside diameter; World Precision Instruments) by using a P97 Micropipette puller (Sutter Instruments). Resistance of the resulting electrodes was between 1.5 and 6.5 MΩ (mean = 3.8 ± 0.1 MΩ in 339 recordings) when filled with standard internal solution. The bath was connected to ground via 140 mM sodium chloride agar bridges. Currents were sampled at 20 or 50 kHz and filtered at 10 kHz by using an EPC9 amplifier (HEKA) controlled with HEKA's Pulse software. Leak and capacitive currents were subtracted online by use of a −P/5 protocol. Recordings obtained with voltage ramps were leak-corrected by a linear fit to the current recorded between −80 and −50 mV, which was extrapolated to +60 mV and subtracted from the whole recording. Series resistance (R s ) was compensated electronically by up to 90% (mean R s after compensation = 2.7 ± 0.1 MΩ) and continuously monitored throughout the measurements. The charging time-constant after compensation was always ≤100 µs (mean = 38 ± 1 µs), and the maximum uncompensated R s -error at the time of peak current was ≤2.5 mV (mean = 0.82 ± 0.04 mV). All experiments were performed at room temperature, from a holding potential of −80 mV and, unless noted otherwise, in cells with a whole-cell capacitance (C s ) between 5 and 25 pF (mean = 15 ± 1 pF), as estimated from the slow capacitance compensation of the amplifier. Perforated patch recordings were performed by using β-escin, which was dissolved in type I ultrapure water to prepare a 25 mM stock solution, protected from light and stored at −20°C for up to 2 wk. Before the recordings, aliquots of the stock solution were diluted in standard internal solution and vortex-mixed for 1 min to give a final escin concentration of 40 µM. After patch formation, gradual perforation was observed as a progressive increase in the speed and amplitude of capacitive transients during 5-ms voltage-steps to −75 mV. Cells showing signs of spontaneous patch rupture (i.e., sudden increase in the amplitude and speed of capacitive transients) were omitted or used as control recordings to exclude that β-escin itself affected the channel properties or the time course of changes in I Ba .

Recording solutions and drugs
All solutions for electrophysiological experiments were prepared by using type 1 ultrapure water (Milli-Q by Millipore Corporation or Purelab Flex 2 by ELGA Labwater) and, unless noted otherwise, reagents purchased from Sigma-Aldrich. During the recordings, cells were constantly perfused with external solution containing (mM) 120 NaCl, 5 BaCl 2 or CaCl 2 , 5 KCl, 1 MgCl 2 , 20 TEA chloride, 10 glucose, 10 HEP ES, and 0.1 BaEDTA with the pH adjusted to 7.4 by using NaOH and osmolarity of 300-320 mOsm. The solution was filtered through 0.2-µm polyethersulfone membranes and applied to cells at a rate of ∼2-4 ml/min by using a gravity-driven perfusion system controlled by manual precision flow regulators (Sarstedt). The standard intracellular solution was composed of (mM) 130 CsCl, 5 oxaloacetic acid, 5 creatine, 5 pyruvic acid, 10 EGTA, and 10 HEP ES with the pH adjusted to 7.3 by using CsOH and osmolarity of 275-295 mOsm. It was stored at −20°C, thawed on the day of the experiments, filtered through 0.2-µm surfactant-free cellulose acetate membranes (Corning), and kept on ice between the recordings. The liquid junction potential between internal and external solution (calculated by using the JPcalc algorithm in pClamp 10; Molecular Devices) was ∼5.5 mV. Because no correction for the liquid junction potential was done, all voltages shown were actually 5.5 mV more negative.
Nucleotide triphosphates (NaATP, MgATP, and MgGTP) were dissolved in 10 mM HEP ES to prepare 100 mM stock solutions (pH = 7.3), stored at −20°C and diluted in standard internal solution immediately before the recordings. Calmodulin (CaM) was dissolved in type I ultrapure water to prepare a 100 µM stock solution and stored at −20°C. AMP-PNP was used as the lithium salt and dissolved directly in the standard internal solution. Deltamethrin,wortmannin,U73122,genistein,and staurosporine (stauro) were dissolved in DMSO to prepare 2-50 mM stock solutions, protected from light and stored at −20°C. Before the experiments, they were diluted in standard internal solution, and the same amount of DMSO (0.1-0.25% vol/vol) without drug was added to the internal solution used for parallel control recordings. To account for the short half-life in aqueous media, all solutions containing wortmannin were discarded after a maximum of 20 min after its dilution into standard internal or external solution, respectively. Cytosolic extract was prepared on the day of the experiments by freeze/thaw lysis of ∼2.1 × 10 7 cells in 1.5 ml ice-cold pipette solution. After centrifugation at 0°C for 5 min, the supernatant was filtered through a 0.2-µm surfactant-free cellulose membrane (Corning), an aliquot was removed for quantification of the protein concentration, and the rest was immediately used for the recordings.

Whole-cell protocols
Unless noted otherwise, time-course recordings were performed by repetitive application at 0.03 Hz of a 30-ms test pulse to +10 mV followed by 10-ms repolarization at −50 mV to record well resolved tail-currents. In some experiments, a 50-ms voltage ramp from −80 mV to +60 mV (2.8 V/s) was used instead of the voltage steps to monitor changes in the quasi steady-state voltage dependence. Because recordings with drugs added to the internal solution could not be used as their own control, we always performed parallel control recordings with standard internal solution. To study changes in current immediately after establishing the whole-cell configuration, cells were clamped at the holding potential of −80 mV before disruption of the patch. After patch rupture, capacitance was quickly compensated by using the auto function of the amplifier, and stimulation started after a delay of no more than 10 s. Because initial current amplitudes were small and several seconds were often required for R s to stabilize completely, the first response was always recorded without R s compensation. We then stopped stimulation, adjusted the R s compensation of the amplifier to achieve maximal compensation without ringing, and restarted the stimulation protocol. To reduce variation introduced by differences in the time required for stabilization of R s , initial values (I start ) for statistical comparison were always calculated as the average of the first two responses, which also provided a compromise between the lack of R s compensation during acquisition of the first response and the time that had already evolved when the second response was recorded. To construct steady-state current-voltage (IV) relationships, peak currents recorded with a protocol consisting of 25-ms test pulses to potentials between −80 mV and +60 mV (10-mV increments at 0.1 Hz) were normalized by the maximum current amplitude at time 0 and plotted as a function of the test-pulse potential. Instantaneous IV (IIV) relationships were obtained with a protocol consisting of a fixed 10-ms prepulse to +60 mV followed by 40-ms test pulses to potentials between −80 mV and +60 mV (10-mV increments at 0.1 Hz). Instantaneous (tail) current amplitudes recorded during the test pulses were normalized by the maximum tail current amplitude at time 0 and plotted as a function of the prepulse potential. The voltage dependence of activation was assessed by a protocol consisting of 25-ms prepulses to potentials between −80 mV and +60 mV (10-mV increments at 0.1 Hz) followed by a fixed 10-ms test pulse to −50 mV. Tail-current amplitudes recorded during the test pulse were normalized to the maximum tail-current amplitude and plotted as a function of the prepulse potential to construct isochronous activation curves. The fraction of channels available for activation from different holding potentials was assessed by a protocol consisting of 3-s conditioning prepulses at potentials between −120 mV and +10 mV (10-mV increments at 0.1 Hz) followed by a fixed 35-ms test pulse to +10 mV. To construct prepulse inactivation curves, peak current amplitudes recorded during the test pulse were normalized by the maximum amplitude and plotted as a function of the prepulse potential.

Data analysis and statistics
Leak-subtracted current traces were directly analyzed with PulseFit (HEKA) or exported for further processing with Microsoft Excel 2010 and OriginLab Pro (version 9; OriginLab).
Time-course recordings were quantified in terms of the current increase during run-up, the duration of run-up, and the residual current after 7 min of run-down, as detailed in the results section. To compare the effect of various test solutions, each of these parameters was also normalized by the mean value observed in parallel control recordings according to Eq. 1: where p t is the value of parameter p measured in a cell with test solution t, ¯ p c is the mean value of p in parallel control recordings, and Δp t is the normalized value of p, which should provide an estimate for the amount of change over control. IV relationships were fitted with a combined Ohm-Boltzmann equation (Eq. 2): where I is the (normalized) peak current density measured at the test potential V m , V rev is the apparent reversal potential, g IV is the maximum slope conductance, V 0.5 is the voltage eliciting half-maximal inward currents, and k is the slope factor. Isochronous activation and prepulse inactivation curves were fitted with single Boltzmann equations (Eq. 3): where I/I max is the normalized current at the prepulse potential V m , V 0.5 is the voltage of half-maximal activation (V 0.5act ) or inactivation (V 0.5inact ), k is the activation (k act ) or inactivation (k inact ) slope factor, and A 1 and A 2 are the initial and final values, respectively. All fits were performed by using the Levenberg-Marquardt least-squares algorithm, and the goodness of fit was judged based on residual plots and adjusted χ 2 values.
Smooth curves in the figures represent fits to average data whereas values given in the text are average data from fits to individual measurements. Values in the text and figures are expressed as mean ± SEM based on the number of independent experiments. Statistical significance was assessed with Origin-Lab Pro 9 by using a repeated-measures ANO VA followed by Bonferroni's post hoc analysis when comparing mean values from the same cells or a one-way ANO VA followed by Bonferroni's post hoc analysis when comparing multiple independent mean values. Homogeneity of variances between groups was tested by using Levene's test for equality of variances on the squared deviations. In the case of heteroscedastic data (P < 0.05 in Levene's test and ratio of largest-to-smallest variance ≥4), statistical significance was assessed with Minitab (version 17; Minitab Inc.) by using Welch's ANO VA and the Games-Howell multiple-comparison method. The secondary Ca v 2.3 channel structure in Fig. 14 was visualized by using the web-based tool Protter for interactive protein feature visualization (available at http:// wlab .ethz .ch/ protter/ start/ ; Omasits et al., 2014). Phosphorylation sites in the same figure were predicted at the highest threshold with the Group-based Prediction Software for Prediction of Kinase-specific Phosphorylation Sites 3.0 (available at http:// gps .biocuckoo .org; Xue et al., 2008).

Results
Evolution and run-down of cloned Ca v 2.3 channel currents Fig. 1 summarizes changes over time in macroscopic Ca v 2.3 channel currents carried by 5 mM Ba 2+ and evoked by voltage-steps to 10 mV in dialyzed HEK-293 cells. Peak current amplitudes recorded immediately after establishing the whole-cell configuration (I start ) were small and tended to increase for several minutes (run-up) until they reached a maximum value (I max ). This was invariably followed by a progressive but somewhat slower current decline (run-down), so that the response that remained 7 min after complete run-up (I 7min ) amounted to approximately half of the maximum current amplitude (Fig. 1, A and B). We quantified the time course of changes by estimating (1) the time required after patch rupture for currents to reach I max (= time to I max ), (2) the ratio between I max and I start (= increase), and (3) the ratio between I 7min and I max (= remaining I Ba ). Under standard recording conditions ( Fig. 1), with mean R s and C s values of 6 ± 1 MΩ and 14 ± 1 pF, respectively (n = 10), run-up took 4 ± 1 min and was associated with a 2.7 ± 0.2-fold increase of peak I Ba at 10 mV, whereas the residual current amplitude after partial run-down amounted to 42 ± 4% of its maximum value.
As illustrated by inspection of scaled current traces ( Fig. 1 C), the initial increase of currents was paralleled by a decrease of fractional inactivation during the 30-ms test-pulses from 53 ± 4% immediately after establishing the whole-cell configuration to 40 ± 4% (P < 0.001) after complete run-up. On the other hand, run-down tended to increase inactivation, although quantification of this effect was often confounded by the reduced signal-tonoise ratio due to current decline (but see also Fig. 13 E).
The general time course of changes was very similar among cells with different peak current densities, not attributable to voltage loss or R s variation ( Fig. 1 B) and unaffected by inclusion of two 3-min resting periods without stimulation (not depicted).

Factors involved in run-up and run-down
Increased mechanical tension associated with fluid flow has been shown to cause run-up and functional alterations in some other VGCCs (Peng et al., 2005;Park et al., 2007), but the time-dependent (B) Same data as in A but normalized to the maximum peak current amplitude (I max ). Gray lines in the background indicate the time course of changes in individual recordings. Also shown above is the lack of spontaneous changes in R s over the time course of the recordings. (C) Same current traces as in A but scaled to their maximum amplitude to visualize changes in their shape. Values are expressed as mean ± SEM. changes in Ca v 2.3 channel current were still observed when recordings were performed under static (i.e., without perfusion) conditions (n = 3; not depicted). As would be expected for a process that involves diffusion-controlled depletion of metabolic substrates or other cytosolic factors on the other hand, the time course but not magnitude of changes in I Ba depended on the absolute values of R s (i.e., cell-pipette coupling) and C s (i.e., cell size), so that combined use of smaller pipette tip diameters (R s = 12 ± 1 MΩ) and larger cells (C s = 29 ± 6 pF) resulted in a significant slowing of run-up and run-down compared with standard recording conditions (Fig. 2, A-D). Unless noted otherwise, the following experiments were all performed under standard recording conditions to facilitate fast and reproducible cell dialysis.
As shown in Fig. 2 E, the characteristic sequence of changes was also observed when recordings were performed with 5 mM Ca 2+ as the charge carrier ( Fig. 2 E), although run-up was significantly faster (Fig. 2 F) and appeared to be more complete when compared with parallel recordings with 5 mM Ba 2+ . Moreover, I start (but not I max or I 7min ) was significantly larger in recordings performed with Ca 2+ , suggesting that run-up but not run-down may be initiated or facilitated by a Ca 2+ -dependent mechanism.
Because changes in the ionic conditions during cell dialysis could affect currents by altering the driving force, we also examined the quasi-steady-state voltage dependence of I Ba with a 50-ms voltage-ramp from −80 to 60 mV ( Fig. 2 G). Run-up was similar over the whole voltage range and not associated with changes in the apparent reversal potential (V rev ), so that initial (I start , blue solid line) and run-up (I max , black solid line) currents overlapped almost completely when scaled to their maximum amplitude ( Fig. 2 G, inset). Currents recorded after partial rundown (I 7min , orange dotted line) still had the same V rev , but were shifted to more negative stimulus voltages, suggesting that their decline is not the result of a change in driving force but influenced by the test potential. with R s = 6 ± 1 MΩ and C s = 14 ± 1 pF; same data as in Fig. 1) or with high-resistance pipettes (R s = 12 ± 1 MΩ) in large cells (C s = 29 ± 6 pF) to retard cytosolic dilution (high C s , n = 6). (B) Comparison of the run-up duration, determined as the time required for currents to reach their maximum amplitude (same cells as in A). (C) Comparison of the residual current after partial run-down, determined as the ratio between current amplitudes after 7 min of run-down and at time 0 (same cells as in A). (D) Comparison of the current increase during run-up, determined as the ratio between maximum and initial current amplitude (same cells as in A). (E) Comparison of time-course recordings performed with 5 mM Ba 2+ (n = 5) or Ca 2+ (n = 5) as the charge carrier. Dotted lines are extrapolated single exponential fits to the evolution of currents during run-up, which yielded the time-constant indicated above. (F) Comparison of the run-up duration observed with Ba 2+ or Ca 2+ as the charge carrier (same cells as in E). (G) Mean current traces evoked by a 50-ms voltage ramp from −80 mV to 60 mV immediately after establishing the whole-cell configuration (I start , blue solid line), at the time of maximum I Ba (I max , black solid line) and after 7 min of run-down (I 7min , orange broken line; n = 7). Inset: Same current traces but scaled to their maximum value to illustrate the lack of changes in position and shape during run-up and subsequent left shift of the curve during run-down. **, P < 0.01; ***, P < 0.001 vs. Ctrl in B and C or vs. Ba 2+ in F (one-way ANO VA with Bonferroni post-hoc correction). Values are expressed as mean ± SEM.

Run-down involves separable changes in gating and maximum conductance
The macroscopic conductance of a uniform population of voltage-gated channels can be approximated by the product of (1) the voltage-and time-dependent channel open probability (P O ), (2) the holding potential-dependent availability for activation (P F ), and (3) the voltage-independent maximum macroscopic conductance (G max ), which depends on single-channel conductance (γ), the total number of channels (N), and the maximum values of P O (P O,max ) and P F (P F,max ). In this framework, run-down could involve reduced activation, increased inactivation, and/or an intrinsically voltage-dependent decrease of G max . In a first attempt to distinguish between these possibilities, we compared steady-state (IV) and instantaneous (IIV) current-voltage relationships, determined at the onset of run-down and in 5-min intervals thereafter (Fig. 3, A-C). Because IIV currents were recorded immediately after a brief prepulse to open most channels available for activation, they should be little affected by changes in the voltage-dependence of P o . Fig. 3 D plots the fraction of IV and IIV currents that remained after partial run-down for 5 min as a function of the test potential. The decline of steady-state currents increased with voltage but reached voltage-independent values at positive test potentials (where P O approaches P O,max ), which is consistent with the shift of ramp-evoked currents (Fig. 2 G). When measured with the IIV protocol on the other hand, run-down was about the same at different test potentials and similar to IV-current decline at depolarized voltages ( Fig. 3 D). The same pattern was observed at the later time points examined, so that the kinetics of IIV-current run-down were well represented by the exponential decline in slope conductance between −80 and 0 mV (τ = 7 ± 2 min, g IIV in Fig. 3 E). The time course of IV-current decline was markedly delayed during weak depolarization but similar when it was measured at sufficiently positive test potentials (τ = 9 ± 1 min) or in terms of the maximum slope conductance (τ = 10 ± 2 min, Figure 3. Voltage dependence of steady-state and instantaneous current decline. (A) Mean steady-state (IV) currents (n = 6) recorded at the indicated test potentials before (black lines) and after 5 min of run-down (orange lines). To allow for comparison of the relative change at different test potentials, they have been normalized to the peak current amplitude observed at each voltage before run-down. (B) Mean instantaneous (IIV) currents recorded from the same cells as in A before (black lines) and after 5 min of run-down (orange lines). To allow for comparison of the relative change at different test potentials, they have been normalized as in A. (C) Comparison of steady-state IV (squares) and IIV (circles) relationships, determined immediately after complete run-up (open symbols) and after 5 min of run-down (orange symbols; same cells as in A and B). (D) Voltage dependence of the fraction of IV (squares) and IIV (circles) current remaining after 5 min of run-down, determined as the ratio between current amplitudes after 5 min of run-down and current amplitudes at time 0 (same cells as in A-C). (E) Mean changes over time in the fraction of IIV current recorded at −20 mV (open circles with dotted line) and +20 mV (black circles with solid line) and in the slope conductance (g IIV ) between −80 and 0 mV (same cells as in A-D). (F) Mean changes over time in the fraction of IV current recorded at −20 mV (open squares with dotted line) and +20 mV (black squares with solid line) and in the maximum slope conductance (g IV ) determined from IV relationships (same cells as in A-E). Values are expressed as mean ± SEM. g IV in Fig. 3 F). Based on these findings, the decrease of macroscopic conductance by itself was independent of the test-pulse potential and had an approximately exponential time course, so the apparent voltage-dependence must have derived from changes in the voltage dependence of P O , which (partly) counteracted IV-current decline and even caused some transient stimulation at negative test potentials.

Run-down is paralleled by changes in channel voltage dependence
To assess how changes in the voltage dependence of P O and P F are involved in current decline, we compared IV, activation, and prepulse inactivation curves (for details on the voltage protocols see Materials and methods), determined at different time points after the onset of run-down. Fig. 4 summarizes the results obtained in a total of 49 cells (C slow = 29 ± 2 pF), from which meaningful currents could be recorded for at least 21 min. The shape of IV current lost to run-down ( Fig. 4 A, inset), was essentially identical to the shape at time 0 and contained a prominent ON-gating current component, suggesting that run-down reduced the total number of active channels (i.e., P F at −80 mV, P F,max , and/or N). It was paralleled by progressive but unequal hyperpolarizing shifts of activation and prepulse inactivation, which are illustrated in Fig. 4 (C and D). The half-activation voltage (V 0.5act ) significantly decreased from 0 ± 1 mV at time 0 to −6 ± 1 mV (P < 0.001) after 21 min of run-down, corresponding to a shift by roughly −6 mV. In the same time, the half-inactivation voltage (V 0.5inact ) significantly decreased from −52 ± 1 mV to −69 ± 1 mV (P < 0.001), corresponding to a shift by roughly −17 mV. In both cases, the rate of shift in individual recordings could be well described by linear fits to the changes in half-point as a function of time, with slopes of −0.33 ± 0.02 mV/min (adjusted R 2 = 0.90 ± 0.02) for activation and −0.82 ± 0.03 mV/min (adjusted R 2 = 0.95 ± 0.01) for inactivation. Based on these findings, P F at −80 mV was reduced by ∼5, 12, and 19% after 7, 14, and 21 min of run-down, respectively (Fig. 4 C), which corresponds to a constant rate of roughly 1%/ min. In the same time, g IV declined by ∼25, 47, and 61%, so that reduced availability for activation alone could only explain part of the IV-current decline at positive test potentials (Fig. 4 B).
Together with a complete loss of gating currents during rundown, these findings suggested that the decline in conductance involves additional (holding potential-independent) changes in the number of active channels (i.e., N and/or P F,max ). This was also evident in time-course recordings where the holding potential after partial run-down was made more negative, which led to considerable but incomplete recovery of the current at 10 mV (not depicted).
In addition to the shift, run-down reduced the steepness of both curves in Fig. 4 C, which was reflected in a gradual increase of the activation and inactivation slope factors (k act and k inact ) from 9.0 ± 0.2 and 9.2 ± 0.1 mV/e-fold change at time 0 to 10.5 ± 0.3 (P < 0.001) and 11.1 ± 0.3 (P < 0.001) mV/e-fold change after 21 min of run-down, respectively (Fig. 4 E).
Hydrolysable ATP provides protection from run-down Because one of the most common reasons for "washout" of ionic currents is a decrease in the level of intracellular high-energy compounds or other cytosolic factors, we next tested if different forms and concentrations of nucleotide triphosphates, a cytosolic extract, and/or perforated-patch recordings are effective in altering run-down. To this end, the time course of changes in peak I Ba under various experimental conditions was quantified as described above and compared with parallel control recordings performed with standard internal solution (Figs. 5 and 6). To facilitate comparison of effects between the different experiments, Fig. 7 summarizes mean changes expressed relative to the corresponding control recordings (i.e., Δ Increase, Δ Time to I max , and Δ Remaining I Ba ), which were determined by subtracting from each individual value with the indicated test solutions the mean value determined in parallel control recordings (for details see Materials and methods). As can be seen in Fig. 5, addition of 1-5 mM MgATP to the pipette solution significantly increased the current fraction that remained after 7 min of rundown compared with parallel control recordings (Fig. 5, A and B) and produced a concentration-dependent slowing of run-up (Fig. 5, A and C). These effects were not attributable to changes in intracellular free Mg 2+ (∼0.4 mM with 5 mM MgATP) because addition of 1 mM MgCl 2 alone (∼0.5 mM free Mg 2+ ) to the standard internal solution affected neither run-up nor run-down (Fig. 5 E). Moreover, with a pipette solution containing 5 mM NaATP and no added free Mg 2+ , there was still a significant slowing of run-up and a significant increase of the current fraction remaining after 7 min of run-down (Fig. 7, A and C).
ATP protection from run-down was about the same with different charge carriers (i.e., Ba 2+ vs. Ca 2+ ) and not altered when the nucleotide was combined with 3 µM CaM (Fig. 7 C). Supplementation of the pipette solution with 0.3 mM MgGTP (Fig. 5 F) or 5 mM of the nonhydrolyzable ATP-analogue AMP-PNP (Fig. 5 G) on the other hand affected neither run-up nor run-down (Fig. 7). Together, these findings argue against a role of direct nucleotide binding and indicate a requirement for ATP-hydrolysis and transfer of the phosphate group.
Note also that (with Ba 2+ as the charge carrier) none of the manipulations tested significantly altered the magnitude of run-up (Figs. 5 B and 7 B) or the absolute values of I max (not depicted), suggesting that ATP can slow but not prevent the processes underlying run-up. The apparent increase in the magnitude of run-up by ATP observed with Ca 2+ as the charge carrier (Fig. 7 B) was also not related to a more pronounced up-regulation of I Ca per se because absolute values of I max were the same as in parallel control recordings (not depicted). Although there was a tendency for I start to be smaller in recordings with ATP, this effect could hardly be ascribed to an action of the nucleotide. Because the faster run-up kinetics in Ca 2+ hampered accurate determination of I start , it might instead have resulted from small differences between groups in the time required for R s to stabilize after patch-rupture.

ATP protection is reproduced by perforated-patch recordings
In principle, exogenous ATP might counteract run-down by preventing a decrease of cytosolic nucleotide levels or by increasing them above the normal value in intact cells, thereby stimulating the channels through an unrelated mechanism. To distinguish between these two possibilities, we performed perforatedpatch recordings with β-escin, diluted into the same internal recording solution as in ruptured-patch experiments. ATP-diffusion through β-escin pores has been demonstrated, but perforating efficiency is concentration and time dependent, so washout should be considerably slower than in ruptured-patch recordings (Arnould et al., 1996;Fan and Palade, 1998;Fu et al., 2003). As illustrated in Fig. 6 A and summarized in Fig. 7, the time course of changes during perforated recordings was very similar to that observed in ruptured recordings with ATP. Importantly, and consistent with a role of ATP-depletion for the current decline, perforated recordings were almost as effective in reducing run-down as provision of ATP in ruptured recordings. Moreover, the protective effects of perforated recordings and ATP were not additive, so that provision of 5 mM MgATP in perforated recordings produced the same effects as in ruptured recordings (Figs. 6 B and 7 C). Collectively, these findings indicate that both, perforated recording and/or provision of ATP reduced run-down by preventing depletion of the nucleotide during the recordings. This is in contrast to the effects on run-up duration, which were much more pronounced in perforated recordings and further increased in the presence of ATP (Figs. 6 C and 7 A). Moreover, because our measurements were started only after R s had reached values ≤15 MΩ, we most likely missed part of the run-up process, so these results may still underestimate the true slowing of run-up in perforated recordings. Thus, the initial but not maximum peak current density was significantly larger in perforated recordings (not depicted), and this was reflected in a reduced magnitude of run-up (Figs. 6 D and 7 B). Together, these observations point to the involvement of additional cytosolic factors during run-up, which are more effectively retained in perforated-patch recordings. However, a cytosolic extract prepared in standard internal solution (4.32 mg protein/ml) was ineffective in altering run-up or rundown (Fig. 7), possibly because these factors depend on the additional presence of ATP.
ATP stabilizes channel gating and maximum conductance All the above findings supported the assumption that Ca v 2.3 channel run-down and, to some extent, run-up in dialyzed cells are related to a depletion of cytosolic ATP. Because it was of interest how ATP affects the different alterations associated with run-down, we reexamined the changes in channel voltage dependence during current decline with a pipette solution supplemented with 5 mM MgATP. As illustrated in Fig. 8 A, inclusion of ATP completely abolished the early phase of current decline, so that on average, g IV after 7 min of run-down amounted to 97 ± 7% of its initial value (n = 6). In addition, the stabilizing action of ATP was associated with an almost complete prevention of time-dependent changes in channel voltage dependence and sensitivity (Fig. 8, B-E), suggesting that basal ATP-dependent modulation alters channel-gating behavior and is required for maintaining it in a functional state.
The effects of ATP are not related to lipid kinasemediated PIP 2 synthesis Because run-down of several neuronal VGCCs and its reversal by MgATP have been linked to depletion and resynthesis of membrane PIP 2 , respectively (Wu et al., 2002;Suh et al., 2010), we next examined the effects of wortmannin (WM), a potent and Figure 7. Overview of changes in run-up and run-down observed under various experimental conditions. Data are from the same cells as in Figs. 5 and 6 with additional data from 7 recordings with cytosolic extract (Cytosol) and 6 parallel control recordings, 9 recordings with 5 mM NaATP and 7 parallel control recordings, and 13 recordings performed with Ca 2+ as the charge carrier and 5 mM MgATP in the presence or absence of 3 µM CaM plus 5 parallel control recordings. For comparison between the different experimental conditions, all values obtained with a given test solution were normalized by subtracting the mean value observed in parallel control recordings and then averaged, so the derived parameters provide a measure for the mean change relative to control (for details see Materials and methods). Symbols for statistical significance refer to the comparison of normalized values between different experimental conditions. (A) Comparison of mean changes relative to parallel control recordings in the duration of run-up (Δ Time to I max ). (B) Comparison of mean changes relative to parallel control recordings in the magnitude of run-up (Δ Increase).
(C) Comparison of mean changes relative to parallel control recordings in the residual current after 7 min of run-down (Δ Remaining I Ba ). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANO VA with Bonferroni post hoc correction in A and C or Welch's ANO VA with the Games-Howell multiple-comparison method in B). Values are expressed as mean ± SEM.
irreversible lipid kinase inhibitor (Wipf and Halter, 2005). When MgATP in the pipette solution was combined with 10 µM WM (ATP+WM in Fig. 9, A-D), it still significantly increased the current fraction remaining after 7 min of run-down (Fig. 9 B) and markedly slowed run-up (Fig. 9 C). Relative to recordings with MgATP alone, WM actually tended to enhance the protective effects ( Fig. 10 C), suggesting that it increased ATP availability because of reduced consumption by lipid kinases. Likewise, pretreatment of cells by incubation in extracellular solution containing 10 µM WM for 15 min (ATP+WM PT in Fig. 9, A-D) did not significantly impair the stabilizing action of MgATP (Fig. 9 B) nor did it alter the time course of changes observed in parallel control recordings. Unlike acute treatment (i.e., ATP+WM) however, WM pretreatment markedly enhanced the ATP-induced slowing of run-up in a subset of cells ( Fig. 9 C), so that on average, the latter effect was stronger but also more variable (Fig. 10 A). A similar modification of ATP-induced changes in the duration of run-up was observed in only one of seven recordings with ATP+WM ( Fig. 9 C), suggesting that PIP 2 depletion rather than acute inhibition of its resynthesis may be required to modify ATP-effects on run-up. Brief application of WM inhibits PIP 2 replenishment without affecting PIP 2 hydrolysis (Zhang et al., 2003), so the variable effectiveness of WM pretreatment might have been related to differences in lipid turnover and degree of actual PIP 2 depletion between cells. Regardless of the exact effects on run-up however, the protection by ATP against run-down was clearly unaffected by WM, suggesting that it was not related to lipid kinase-mediated mechanisms and arguing against a role of PIP 2 depletion for Ca v 2.3 channel run-down in our system.
Another line of reasoning has been that not PIP 2 depletion itself, but rather accumulation of one of its fatty acid cleavage products, arachidonic acid (AA), is responsible for rundown of some neuronal VGCCs (Liu et al., 2001(Liu et al., , 2006Liu and Rittenhouse, 2003). However, neither 1 mg/ml of the AA-scavenger BSA (Fig. 9 E) nor 5 µM of the phospholipase C inhibitor U73122 (Fig. 9 F) affected the time course of run-up or rundown when they were added to the (ATP-free) internal solution (Fig. 10, A-C).
The effects of ATP depend on protein kinase-mediated phosphorylation We next examined the role of changes in protein phosphorylation and dephosphorylation, which may result from ATP-depletion and have been implicated in the run-down of L-type high-voltage-activated Ca 2+ channels (Armstrong and Eckert, 1987;Hilgemann, 1997). As summarized in Fig. 10 (A-C), the effects of MgATP were well preserved and again even somewhat enhanced when it was combined with 100 µM of the protein tyrosine kinase inhibitor genistein (GS). Combination with 10 µM of the broad-spectrum serine/threonine kinase inhibitor stauro on the other hand completely abolished MgATP effects on the duration of run-up, so the time to I max with MgATP+stauro was not significantly different from the time course in parallel control recordings (Fig. 11, A-C; and Fig. 10 A). Moreover, although MgATP in the presence of stauro still significantly increased the fraction of current remaining after 7 min of rundown relative to parallel control recordings (Fig. 11, A and B), the net effect was significantly reduced when compared with that observed in other recordings with MgATP. Thus, expressed relative to the corresponding control recordings, MgATP+stauro was significantly less effective in increasing the current fraction after 7 min of run-down than MgATP alone, MgATP+WM, or MgAT-P+GS (Fig. 10 C).
There was no evident effect of stauro in the absence of ATP (Fig. 11, A-C), indicating that it acted by reducing ATP-protection from run-down. From these findings it follows that the ATP-induced slowing of run-up and most but not all the protection from run-down must have involved protein kinase-mediated phosphorylation of serine/threonine residues. On the other hand, run-down in the absence of ATP might reflect a progressive decrease of channel phosphorylation because of changes in the balance between constitutive de-and rephosphorylation. To test this assumption, we examined the effects of a sustained (30-60min) incubation of cells in extracellular solution containing 1 µM stauro, which should mimic run-down by reducing basal protein phosphorylation. Consistent with (partial) run-down of the channels before establishment of the recordings, stauro pretreatment increased the number of cells lacking macroscopic currents and significantly reduced both I start and I max in the remaining cells (Fig. 11, D-F). Interestingly, it also significantly increased the magnitude and duration of run-up (Fig. 11, G-I), although the latter effect may have been related to the appearance of a plateau phase with little change in current amplitudes for several minutes. Subsequently, run-down proceeded with a normal time course, so the decrease of currents after 7 min was essentially the same as in parallel control recordings.
To assess how these effects were related to the ATP-sensitive gating changes, we also examined the effects of stauro pretreatment on channel voltage dependence (Fig. 12). As illustrated in Fig. 12 (B and C), activation and prepulse inactivation curves recorded in pretreated cells before the onset of run-down showed a significant shift to more negative test potentials when compared with the results obtained in untreated cells, although the voltage-sensitivity (i.e., k act and k inact ) was similar (not depicted).
In addition, stauro pretreatment tended to accelerate the voltage shifts during run-down (Fig. 12, D and E), although comparison of the exact time course is confounded by differences in cell size between the experiments, so no firm conclusions can be drawn from this finding. Interestingly however, it also effectively prevented the decrease in activation voltage sensitivity during run-down (Fig. 12 F) without altering the changes in inactivation voltage sensitivity (not depicted), possibly pointing to the involvement of multiple mechanisms at distinct sites.
Because the serine/threonine phosphatase calcineurin has been shown to interact with neuronal VGCCs (Chad and Eckert, 1986;Fomina and Levitan, 1997) and implicated in the run-down of certain K + channels (Horváth et al., 2002), we also tested the effects of pretreating cells with a 60-min incubation in extracellular solution containing 10 µM of the membrane-permeable calcineurin-inhibitor deltamethrin (DM). However, the time course of changes in I Ba observed in DM-pretreated cells (recorded with ATP-free pipette solution) was not significantly different from that in parallel control recordings (Fig. 10). Run-up may involve activation of Leu-sensitive proteases Finally, we performed experiments with Leu, a protease inhibitor that has been shown to prevent an irreversible ATP-resistant component of run-down in L-type VGCCs (Chad and Eckert, 1986;Elhamdani et al., 1994). Unexpectedly, and in contrast to all other manipulations tested, MgATP+100 µM Leu significantly decreased the magnitude of run-up (Fig. 13, A-C), so that I start was similar but I max was approximately half of the value observed in parallel control recordings. In addition, Leu not only abolished the MgATP-dependent slowing of run-up but actually produced a significant decrease of the time to I max when it was combined with ATP ( Fig. 13 D). Apart from altering the time course and net increase of currents, ATP+Leu reduced cell-to-cell variability in the magnitude of run-up (variance = 0.89 in control recordings vs. 0.16 with MgATP+Leu, P < 0.05) and diminished the initial slowing of inactivation (Fig. 13 C), indicating that both processes may be related to activation of Leu-sensitive proteases and influenced by variable endogenous protease and/or protease inhibitor levels.
Curiously, Leu also suppressed the protective effect of MgATP against run-down (Fig. 13 A), so that expressed relative to the corresponding control recordings, MgATP+Leu was significantly less effective against rundown than MgATP alone, MgATP+WM, MgATP after WM pretreatment, or MgATP+GS (Fig. 10 C). When the same concentration of Leu (100 µM) was used in the absence of MgATP, it had similar but quantitatively much less marked effects on run-up (Fig. 13, A-D) and no effect on the degree of run-down ( Fig. 10 C) or the inactivation changes during run-up (Fig. 13 E).

Discussion
In the present study, we used conventional and perforated-patch-clamp recordings in a recombinant expression system to assess changes in Ca v 2.3 channel currents during cell dialysis.
Our findings recapitulate studies about their native counterparts and show that dialysis with ATP-free internal solutions produces a characteristic sequence of run-up and run-down, the time-course of which depends on R s , cell size, and recording configuration. The exponential decline in conductance during run-down by itself was voltage independent but paralleled by a progressive shift of channel voltage dependence to more negative test potentials. The voltage-shift reduced channel availability at the holding potential (i.e., P F ) but proceeded at a constant rate and could only partly account for the complete loss of gating currents during run-down. Therefore, most of the decline in conductance was related to changes in the total number of functional channels (i.e., N and/or P F,max ) and possibly to other factors (i.e., decrease of γ or P O,max ). Run-down and all the associated Figure 10. Overview of changes in run-up and run-down observed under various experimental conditions. Data are from the same cells as in Figs. 9, 11, and 13 with additional data from six recordings with 5 mM MgATP + 100 µM GS (+GS) and four parallel control recordings and three recordings after a 60-min pretreatment with 10 µM DM and five parallel control recordings. For comparison between the different experimental conditions, all values obtained with a given test solution were normalized as described in Fig. 7 and Materials and methods. Symbols for statistical significance refer to the comparison of normalized values between different experimental conditions. (A) Comparison of mean changes relative to parallel control recordings in the duration of run-up (Δ Time to I max ). (B) Comparison of mean changes relative to parallel control recordings in the magnitude of run-up (Δ Increase). (C) Comparison of mean changes relative to parallel control recordings in the residual current after 7 min of run-down (Δ Remaining I Ba ). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANO VA with Bonferroni post hoc correction in A and C or Welch's ANO VA with the Games-Howell multiple-comparison method in B). Values are expressed as mean ± SEM. biophysical changes could be slowed or prevented by provision of ATP, and this protective action was almost completely abolished by inhibition of serine/threonine kinases. Protein kinase inhibition also mimicked the effects of run-down in intact cells, so it reduced overall peak current densities and hyperpolarized the voltage dependence of gating relative to untreated cells. The effects of ATP could be replicated neither by a nonhydrolyzable ATP analogue nor by GTP, which argues against a role of direct nucleotide binding or G-protein interactions but is consistent with the reported selectivity of protein kinases for ATP (Becher et al., 2013). Finally, run-down was not influenced by dialysis with a PLC inhibitor or the AA scavenger BSA, and the effects of ATP were unaffected by inhibition of lipid kinases, suggesting that run-down was not related to PIP 2 hydrolysis, or accumulation of its cleavage product AA. Based on these findings, we conclude that (1) ATP-protects from Ca v 2.3 channel run-down by maintaining phosphorylation of serine/threonine residues on the channels themselves or an associated protein, (2) constitutive dephosphorylation of these sites in dialyzed cells affects channel gating and reduces the total number of functional channels, and (3) (de-)phosphorylation of at least some of the sites can also regulate channel function in intact cells. In addition, some of our findings point to a role of Leu-sensitive proteases for Ca v 2.3 channel up-regulation during run-up and for the effects of protein phosphorylation on run-down.
Comparison with run-down in other voltagegated Ca 2+ channels Since its first description, Ca 2+ channel run-down has been consistently linked to diffusion-controlled dilution of ATP and other cytosolic components, but the underlying processes seem to differ among channels. ATP-induced protection from L-type Ca 2+ channel run-down involves several interrelated processes, which may include changes in lipid turnover (Wu et al., 2002;Kaur et al., 2015), direct nucleotide binding , and phosphorylation of the channels by PKA, CaMKII and possibly other kinases (Wang et al., 2009;Xu et al., 2016). The latter counteracts constitutive dephosphorylation by opposing protein phosphatases and has been proposed to stabilize conformations that can be reprimed by voltage, CaM, or calpastatin and/or are more resistant to proteolytic degradation (Chad et al., 1987;Wang et al., 2009;Sun et al., 2014). This is in contrast to the situation Figure 11. ATP protection from run-down involves serine/threonine phosphorylation. (A) Mean data from 10 recordings with 5 mM MgATP and 10 µM stauro (ATP+Stauro), 4 recordings with 10 µM stauro only (Stauro), and 12 parallel control recordings (Ctrl). (B) Comparison of the residual I Ba after 7 min of run-down in recordings with Stauro or ATP+Stauro and in parallel control recordings (same cells as in A). (C) Comparison of the run-up duration in recordings with Stauro or ATP+Stauro and in parallel control recordings (same cells as in A). (D) Mean data from seven recordings after pretreatment with 1 µM stauro (Stauro PT) and five parallel control recordings (Ctrl). Note that current amplitudes are expressed in pA/pF to illustrate the reduced peak current densities after stauro pretreatment.
(E) Comparison of the initial peak current densities observed after stauro pretreatment and in parallel control recordings (same cells as in D).   in Ca v 2.1 and Ca v 2.2 channels, where run-down has been linked to constitutive hydrolysis of membrane PIP 2 , which can be prevented by ATP through lipid kinase-mediated PIP 2 resynthesis (Wu et al., 2002;Suh et al., 2010). Although we have not directly tested the effects of PIP 2 , our present findings demonstrate that lipid kinases are not involved in ATP-induced protection from Ca v 2.3 channel run-down and that the current decline is not related to accumulation of AA. Together with a previous study, where PIP 2 -depletion had no effect on Ca v 2.3 channels (Suh et al., 2010), these findings argue against a major role of PIP 2 hydrolysis for Ca v 2.3 channel run-down. Instead, ATP appears to maintain Ca v 2.3 channel function through increased phosphorylation of sites on the channels themselves or an associated protein, which may be required to counteract constitutive dephosphorylation. It has been shown that HEK-293 cells contain endogenous kinases and phosphatases, which can regulate the activity of transfected Ca 2+ channels Johnson et al., 1997;Fuller et al., 2010;Aita et al., 2011;Blesneac et al., 2015). In addition, Ca v 2.3 channels are a well-known substrate for phosphorylation by various protein kinases (Fig. 14), at least some of which can be constitutively active in HEK-293 cells Crump et al., 2006). Although no firm conclusions can be drawn with regard to the exact sites or kinases involved in Ca v 2.3 channel maintenance, our findings provide a reliable baseline for further studies. They also raise the question how protein phosphorylation might be involved in the maintenance Ca v 2.1 and Ca v 2.2 channels, which still exhibit significant rundown in the presence of exogenous PIP 2 (Gamper et al., 2004) or with mutations that reduce PIP 2 sensitivity (Zhen et al., 2006). Because there can be cross talk with lipid signaling (Wu et al., 2002) and Figure 14. Phosphorylation sites and potential PEST regions in human Ca v 2.3 channels. The secondary structure was visualized with Protter based on the human protein database entry Q15878, which includes exon 19 and exon 45 encoded stretches of the full-length Ca v 2.3d-splice variant. Predictions were performed at the highest threshold by using the Group-based System Software for Prediction of Kinase-specific Phosphorylation Sites 3.0 and at a threshold score of +5.0 by using epestfind for detection of potential proteolytic cleavage sites, respectively. CAM KI, Ca 2+ /CaM-dependent protein kinase I; CAM KII, Ca 2+ /CaM-dependent protein kinase II; CAM KL, Ca 2+ /CaM-dependent protein kinase-like kinases; MAPK, mitogen-activated protein kinase; SGK, serum and glucocorticoid-regulated kinase 1; ter, terminal. PIP 2 -hydrolysis partially inhibited Ca v 2.3 channels after full activation by PKC (Jeong et al., 2016), it will also be interesting to examine the exact relevance of lipid turnover for this form of regulation.

Molecular mechanisms and potential implications
Very little is known about the molecular and structural mechanisms of Ca 2+ channel run-down, but possibilities that have been considered include disruption of the linkage between voltage sensors and activation gate, entry into a permanent but existing inactivated state, and spontaneous drops in the total number of channels due to internalization or degradation. Our results do not resolve the exact processes underlying Ca v 2.3 channel rundown but do argue against a significant decoupling between voltage-sensors and activation gate because run-down abolished both ionic and gating currents. In addition, Leu was ineffective against run-down and actually reduced the protective effects of ATP, suggesting that the current decline was not directly related to increased proteolytic degradation (but see next section). Together, these findings contrast with studies that L-type-channel gating currents are not diminished by run-down (Hadley and Lederer, 1991;Costantin et al., 1999) and that Leu prevents an irreversible component of run-down in these channels (Chad et al., 1987). We can only speculate that dephosphorylation of Ca v 2.3 channels themselves or an interacting protein induces a conformational change that leads to terminal inactivation and/ or facilitates their removal from the membrane.
More importantly, our findings reveal that part of the current decline can be attributed to a gradual development of inactivation due to changes in channel voltage dependence. Based on the lack of saturation or concurrent changes in V rev , these effects may have been related to changes in the fraction of applied membrane voltage sensed by the channels. Interestingly, and in contrast to the exponential decline in conductance, the gating changes occurred at a constant rate, which is difficult to reconcile in terms of a conformational change. An attractive hypothesis that remains to be substantiated is that progressive dephosphorylation could shift channels among their states in a simple manner, such as altering the internal surface charge. It has been quantitatively demonstrated that the bulk electrostatic effects of dephosphorylation are sufficient to produce hyperpolarizing voltage shifts during run-down of other voltage-gated ion channels Bezanilla, 1990, 1991). Regardless of the exact mechanism, our findings document a close relationship between protein phosphorylation and channel voltage dependence, which may influence the basal features of current carried by cloned Ca v 2.3 channels. Based on previous studies about their native counterparts, it seems reasonable to propose that this is also relevant under physiological conditions. Thus, Ca v 2.3 channels are the third most extensively phosphorylated ion channels in mouse brain (Cerda et al., 2011;Fig. 14), and depolarization of intact hippocampal slices has been shown to induce bulk changes in their phosphorylation state (Hell et al., 1995).
Run-up and the role of calpain-like proteases Unexpectedly, our findings also indicate that Ca v 2.3 channel run-up may involve activation of calpain-like proteases (CLPs) and that it can be slowed but not prevented by ATP through increased protein phosphorylation. The process started immediately upon patch rupture, which can hardly be accounted for by ATP depletion but might reflect the loss of small cytosolic protease inhibitors because it was much slower in perforated recordings. In addition, run-up was observed with Ba 2+ as the charge carrier but significantly faster and more complete with Ca 2+ , which is consistent with studies that show that Ba 2+ can partially substitute for Ca 2+ in activating CLPs (DeMartino and Croall, 1985;McDonald et al., 1994;Seydl et al., 1995). That phosphorylation suppresses most CLPs (Shiraha et al., 2002;Smith et al., 2003), while PIP 2 is well known to be required for activation and may considerably lower their Ca 2+ requirement (Tompa et al., 2001;Leloup et al., 2010), could explain why (1) ATP, but not AMP-PNP or ATP+stauro, slowed run-up and (2) pretreatment with WM (i.e., partial depletion of membrane PIP 2 ) further increased the ATP-induced slowing in some cells. Dialysis or pretreatment with stauro alone had no effect or even increased run-up, suggesting that the process was not related to proteolytic activation of protein kinases. With this in mind and considering its fast onset, we can only speculate that run-up might involve partial proteolysis of the channels themselves and/or associated Ca v β 3 subunits. Both proteins contain several PEST motifs or PEST-like regions in their C and N termini (Fig. 14), and there is convincing evidence for a functional relevance of these sites as potential cleavage sites. For example, deletion of PEST-like regions in the Ca v β 3 subunit increased its half-life, stimulated currents mediated by coexpressed Ca v 2.2 channels, and reduced voltage-dependent inactivation in HEK-293 cells (Sandoval et al., 2006). Likewise, intracellular protease application has been shown to produce strong stimulation of VGCCs and a partial loss of fast voltage-dependent inactivation, which has been linked to C-terminal cleavage of a conserved autoinhibitory region in the pore-forming Ca v α 1 subunit Klöckner et al., 1995;Gao et al., 2001;Mikala et al., 2003). Given that C-terminal truncation of L-, N-and P/Q-type VGCCs by endogenous proteases has been demonstrated before in HEK-293 cells (Kubodera et al., 2003;Gomez-Ospina et al., 2006) and in vivo (Gerhardstein et al., 2000;Abele and Yang, 2012), it seems not so far off that the structural determinants for Ca v 2.3 channel run-up could also be located in this region. With regard to the Leu effects on run-down, it may be important that L-type channels can also be cleaved at PEST regions within the core of the Ca v α 1 subunit, which has been shown to disrupt channel function (Groth et al., 2014;Michailidis et al., 2014). That Ca v 2.3 channels lack such regions might account for the inability of Leu to sustain ATP-induced protection. On the other hand, this cannot explain the puzzling finding that Leu actually impaired the protective effects of ATP on run-down. We can only speculate that the latter might be related to differential phosphorylation of long and short forms or changes in the accessibility of certain sites. Because Leu could also alter the effects of ATP in some other way, further studies will clearly be required to delineate the underlying processes. However, our finding that protein kinase inhibition alone replicated most of the gating changes during run-down in intact cells leads us to conclude that proteolysis is either not required for these effects or that it does also occur in intact cells.

Conclusion
In summary, our findings show that run-down of cloned Ca v 2.3 channels in dialyzed cells is associated with progressive changes in channel voltage-dependence and a decrease in the total number of functional channels, which can be prevented by ATP through maintained protein phosphorylation and replicated by protein kinase inhibition in intact cells. These findings distinguish the process from run-down in other Ca v 2 channels and suggest that one or more sites on the channels themselves or an associated protein must be phosphorylated to maintain them in a functional state. In addition, changes in the phosphorylation of a subset of sites may directly influence channel gating, possibly through bulk electrostatics. Although additional studies will be required to delineate the underlying mechanisms and their relevance in native cells, our results provide a reliable baseline that could stimulate further work on Ca v 2.3 channel regulation by protein kinases, phosphatases, and possibly proteases.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
High numbers of PDCD1 (PD-1)-positive T cells and B2M mutations in microsatellite-unstable colorectal cancer

ABSTRACT DNA mismatch repair (MMR)-deficient cancers accumulate high numbers of coding microsatellite mutations, which lead to the generation of highly immunogenic frameshift peptide (FSP) neoantigens. MMR-deficient cells can grow out to clinically manifest cancers either if they evade immune cell attack or if local T-cells get exhausted. Therefore, a subset of MSI cancer patients responds particularly well to treatment with immune checkpoint inhibitors. We analyzed whether immune evasion in MMR-deficient cancer mediated by loss of HLA class I or II antigens is related to local immune cell activation status. Microsatellites located in Beta2-microglobulin (B2M) and the HLA class II-regulatory genes RFX5 and CIITA were analyzed for mutations in MMR-deficient colorectal cancers (n = 53). The results were related to CD3-positive and PDCD1 (PD-1)-positive T-cell infiltration. PDCD1 (PD-1)-positive T-cell counts were significantly higher in B2M-mutant compared to B2M-wild type tumors (median: 22.2 cells per 0.25 mm2 vs. 2.0 cells per 0.25 mm2, Wilcoxon test p = 0.002). Increasing PDCD1 (PD-1)-positive T-cell infiltration was significantly related to an increased likelihood of B2M mutations (OR = 1.81). HLA class II antigen expression status was significantly associated with enhanced overall T-cell infiltration, but not related to PDCD1 (PD-1)-positive T-cells. These results suggest that immune evasion mediated by B2M mutation-induced loss of HLA class I antigen expression predominantly occurs in an environment of activated PDCD1 (PD-1)-positive T cell infiltration. If B2M mutations interfere with anti-PDCD1 (PD-1)/CD274 (PD-L1) therapy success, we predict that resistance towards anti-PDCD1 (PD-1) therapy may – counterintuitively – be particularly common in patients with MMR-deficient cancers that show high PDCD1 (PD-1)-positive T cell infiltration.

Introduction
Cancers of the microsatellite instability (MSI) phenotype are caused by deficiency of the DNA mismatch repair (MMR) system. MSI cancers are distinguished among other features by their high immunogenicity. This likely results from an exceptionally high mutational load: MMR deficiency leads to the generation of multiple insertion/deletion mutations in coding microsatellites and consequently to mutation-induced frameshift peptide (FSP) neoantigens. 1 MSI is observed in 15% of all colorectal cancers and in a variety of extracolonic malignancies such as endometrial cancer, of which up to 30% are MSI. [2][3][4] MSI cancers can develop sporadically, most frequently through somatic epigenetic inactivation of the MMR gene MLH1. 5 Alternatively, they occur in the context of the hereditary non-polyposis colorectal cancer (HNPCC) or Lynch syndrome that is caused by germline mutations of MMR genes, most frequently MLH1 or MSH2 6 MSI colorectal cancers commonly present with a dense lymphocyte infiltration, typically of an activated and cytotoxic phenotype. 7-10 FSP-specific tumor-infiltrating lymphocytes from MSI cancers in fact retain the potential of lysing MSI tumor cells. 11 Despite the evidence of local anti-tumoral immune responses, MSI tumors often grow out to large and clinically relevant size, which suggests the existence of immuno-suppressive and/or immuno-evasive mechanisms. In fact up to 30% of all MSI colorectal cancers display mutations in the B2M gene with a consecutive loss of HLA class I antigens that prevents recognition as well as killing of B2M-mutant cells by cytotoxic T cells. 12,13 B2M mutations, as a likely mechanism of immune evasion, are thought to provide affected MSI tumor cells with a major selection advantage as it has been reported for other tumor types such as malignant melanoma. 14,15 More recently, an additional mechanism has been proposed to contribute to the immune evasion of MSI tumor cells: loss of functional HLA class II antigen presentation machinery occurs in approximately one third of all MSI CRCs as a consequence of mutations inactivating the HLA class II-regulatory genes RFX5 and CIITA. 16 an alteration that is also associated with increased local T cell infiltration of the tumor. 17 Immune checkpoint molecules such as PDCD1 (PD-1) have gained attention as targets for novel immune therapy approaches. [18][19][20][21][22] The physiological role of PDCD1 (PD-1) is to preserve the equilibrium between co-stimulatory and coinhibitory signals that regulate T cell activity and maintain selftolerance. In tumors, PDCD1 (PD-1) can limit the potency of anti-tumoral immune responses (T cell exhaustion) upon activation by one of its ligands (CD274 [PD-L1], PDCD1LG2 [PD-L2]). 22,23 This may facilitate the outgrowth of even highly antigenic tumors in the presence of tumor-antigen specific immune cells. Antibody-mediated blockade of PDCD1 (PD-1) or CD274 (PD-L1) has recently emerged as a promising novel method of cancer immunotherapy, particularly in MSI cancer patients. [24][25][26][27][28] Although a substantial proportion of approximately half of advanced MSI cancer patients showed objective responses to anti-PDCD1 (PD-1) antibody therapy, even including radiographic complete responses in almost 20%, about one fourth of patients had progressive disease under therapy. 27 So far, markers predicting the success of anti-PDCD1 (PD-1)/CD274 (PD-L1) therapy among MSI cancer patients are not yet established. Similarly, studies are lacking that investigate the relationship of PDCD1 (PD-1)-positive T cell infiltration in MSI cancers with immune evasion phenomena such as alterations of HLA class I or II-mediated antigen presentation 26 Such more comprehensive information about the tumor immune status could potentially be highly relevant in the identification of patients expected to benefit from immune therapy by PDCD1 (PD-1)/CD274 (PD-L1) pathway blockade. 29,30 We therefore analyzed the local infiltration of MSI colorectal cancers by PDCD1 (PD-1) positive T cells and investigated the effect of local PDCD1 (PD-1) positive T cell infiltration on B2M mutation status and HLA class II antigen expression pattern of the respective tumors.

B2M mutation status and HLA class II expression status of MSI colorectal cancers
In order to examine a potential influence of the infiltration of MSI colorectal cancer lesions with immune cells on B2M mutation status and/or HLA class II antigen expression status of the tumors, immunohistochemical staining was performed. Representative staining results are shown in Fig. 1 Percentages are given in brackets

Relation between immune cell infiltration and B2M mutation status
We investigated the association of general tumor lymphocyte infiltration with tumor B2M mutation status and did not observe a statistically significant change in distribution of the intratumoral CD3-positive T cell infiltration with respect to B2M mutation status (median: 101.  Fig. 2b). In a multivariate model, PDCD1 (PD-1)-positive T cell infiltration was identified as the only statistically significant predictor being positively associated with the presence of B2M mutation (odds ratio for doubling of PDCD1 [PD-1]positive T cell counts, OR D 1.69, Table 2). Also after variable selection, PD-1-positive T cell infiltration remained a significant predictor in the model and showed a similar effect (OR D 1.48).

Relation between immune cell infiltration and HLA class II antigen expression
We observed a significantly higher overall T cell infiltration in tumors with higher HLA class II antigen expression (median 150.0 cells per 0.25 mm 2 HLA class II antigen 2 vs. 133.9 cells per 0.25 mm 2 HLA class II antigen 1 vs. 50.8 cells per 0.25 mm 2 HLA class II antigen 0, p < 0.001, Fig. 3a). PDCD1 (PD-1)-positive T cell infiltration also tended to be higher in tumors with higher HLA class II antigen expression; however, the proportion of PDCD1 (PD-1)-positive T cells among all T cells was not significantly related to HLA class II antigen expression (Fig. 3b, In a multivariate model, CD3-positive T cell infiltration was identified as the only statistically significant predictor being positively associated with higher HLA class II antigen expressions (odds ratio for doubling of CD3-positive T cell counts, OR D 2.48, for 'lack of or barely detectable' vs. 'intermediate' as well as for 'intermediate' vs. 'high', Table 3). Also after variable selection, CD3-positive T cell infiltration remained a significant Figure 2. Distribution of specific T cell infiltration data in B2M-mutant (mt) and B2M-wild type (wt) MSI colorectal cancers A There was no statistically significant difference in intratumoral CD3-positive T cell infiltration between B2M-mutant (mt) and B2M-wild type (wt) MSI colorectal cancers B A significantly higher number of PDCD1 (PD-1)positive T cells was observed in B2M-mutant compared to B2M-wild type MSI colorectal cancers. predictor in the model, but with a somewhat smaller effect on the HLA class II antigen expression (OR D 1.88).

Discussion
A central observation of our study is that alterations to tumor antigen presentation molecules are related to the local lymphocyte infiltration patterns. Our results suggest that mutations in the B2M gene, which are the most frequent cause of complete breakdown of HLA class I-mediated antigen presentation in MSI colorectal cancer, are specifically related to a high number of tumor-infiltrating PDCD1 (PD-1)-positive T cells.
In contrast, we did not observe any significant difference of overall T cell counts between B2M-wild type and B2M-mutant tumors. This is in line with several previous studies that did not observe any correlation between immune cell infiltration and B2M mutation status. 17,31 except the study by de Miranda et al. who found evidence of higher counts of certain T cell subsets infiltrating B2M-mutant compared to wild type tumors. 32 However, increasing PDCD1 (PD-1)-positive T cell infiltration was significantly related to an increased likelihood of B2M mutation.
Our results for the first time establish B2M mutation as a consequence of high PDCD1 (PD-1)-positive T cell counts in MSI cancers. Potentially underlying this is the influence of the local immune cell milieu on the phenotype of the emerging tumors, as postulated by the immunoediting model. [33][34][35][36] In an environment of dense infiltration with activated PDCD1 (PD-1)-positive T cells, reflective of a more active local immune milieu and thus a stronger immunoselective pressure, emerging HLA class I-positive tumor cell clones may be eliminated more effectively. This probably favors the outgrowth of poorly immunogenic, B2M-mutant MSI colorectal cancer cells (Fig. 2). These no longer present FSP-derived neoantigens to the immune system via HLA class I antigens. This model is in line with the previous observation by Giannakis et al. 37 who reported an increased likelihood of mutations affecting B2M or other genes involved in HLA class I-mediated antigen presentation in colorectal cancers densely infiltrated by lymphocytes.
In fact, PDCD1 (PD-1)-positive T cell infiltration remained the only significant predictor of B2M mutation status in a multivariate model. No significant relationship between the proportion of PDCD1 (PD-1)-positive T cells and HLA class II antigen expression on tumor cells was observed. However, in concordance with the previously reported association of HLA class II antigen expression with CD4-positive T cell infiltration 17 overall T cell density was significantly increased in tumors with higher HLA class II antigen expression.
Lynch syndrome-associated cancers showed significantly elevated immune cell infiltration compared to sporadic MSI cancers, which is in line with previous findings. 38,39 In addition, we here for the first time report a significantly higher absolute Abbreviations: OR, Odds ratio; CI, confidence interval Figure 3. Distribution of specific T cell infiltration data in tumors with HLA class II antigen expression status high, intermediate or lack of or barely detectable A We observed a significant increase in overall T cell infiltration in tumors with higher HLA class II antigen expression B PDCD1 (PD-1)-positive T cell infiltration tended to increase with higher HLA class II antigen expression, but the difference did not reach statistical significance. and relative amount of PDCD1 (PD-1)-positive T cells in Lynch syndrome vs. sporadic MSI cancer specimens. This suggests that the recurrent exposure of immune cells towards MSIrelated antigens in Lynch syndrome, e.g. through mismatch repair-deficient crypts 1,40,41 may lead to T cell activation and consequently an enhanced number of activated T cells in clinically manifest tumors. The recurrent stimulation of the immune system with MSIinduced FSP neoantigens in Lynch syndrome may thus be the reason for the higher frequency of B2M mutations in Lynch syndrome-associated compared to sporadic MSI colorectal cancers 42 This supports the concept that stimulating the immune system against MSI-related FSP antigens, e.g. by a vaccine 1 may not only delay tumor formation in Lynch syndrome, but also lead to a better outcome, because B2M-mutant cancers have an excellent prognosis after surgical removal. 43,44 The presence of functional HLA class I antigens and therefore the presence of functional B2M is a prerequisite for recognition and killing of tumor cells by cytotoxic CD8positive T cells 45 Therefore, B2M mutations are expected to interfere with the success of immune checkpoint modulator therapy, e.g. using pembrolizumab 14,[46][47][48] as has recently been observed as a mechanism of secondary resistance in a melanoma patient and in MSI cancers. 27,49 Accordingly, our results predict that upfront resistance towards immune checkpoint blockade may be particularly common among MSI cancers with high PDCD1 (PD-1)-positive T cell infiltration, because they commonly show B2M mutationinduced loss of HLA class I antigen presentation. This, somewhat counterintuitively, suggests that MSI cancers with low PDCD1 (PD-1)-positive T cell counts will likely respond better to anti-PDCD1 (PD-1) therapy than tumors with high PDCD1 (PD-1)-positive T cell counts.
B2M mutations have only rarely been observed in MSI colorectal cancers presenting with hematogenous metastasis formation, for example to the liver. 13,43 Possible mechanisms underlying this observation are enhanced NK cell killing of B2M-mutant, HLA class I antigen-negative MSI cancer cells 50 or, alternatively, impaired tumor cell/platelet interaction after loss of HLA class I antigen expression. 51 However, B2M mutations still allow metastasis formation through local or lymphatic tumor cell spread. In fact, peritoneal metastasis through non-hematogenous tumor cell spread is a common manifestation in MSI colorectal cancer of stage M1. 52,53 B2M mutation status and infiltration with activated PDCD1 (PD-1)-positive T cells may be of particular predictive significance for checkpoint blockade in this clinical constellation.
In summary, our results suggest that high amounts of PDCD1 (PD-1)-positive tumor-infiltrating T cells, representing a highly active local immune milieu, may favor the outgrowth of emerging B2M-mutant, HLA class I-negative tumor cell clones. This is in line with the hypothesis that B2M mutations in MSI cancer occur as a direct result of DNA mismatch repair deficiency-induced mutations and subsequent immunoselective pressure. It is strongly suggested that all future clinical trials using PDCD1 (PD-1)/ CD274 (PD-L1) checkpoint modulators should account for B2M mutation status and immune cell infiltration of the primary tumor and all available metastatic lesions as potential predictors of therapy efficacy.

Patients and tumor specimens
Tumor samples represent a consecutive series of MSI colorectal cancers recorded in the Department of Applied Tumor Biology, Institute of Pathology, University Hospital Heidelberg as part of the German HNPCC Consortium. Informed consent was obtained from all patients included in this study. The Ethics Committee of the University of Heidelberg approved the study. None of these patients underwent neoadjuvant radio-and/or chemotherapy prior to surgery.
Microsatellite instability status was determined using the Bethesda standard marker panel (BAT25, BAT26, D2S123, D5S346 and D17S250/Mfd15) and CAT25 as described previously. 54,55 Tumors from patients with a germline mutation of the DNA mismatch repair genes were classified as 'hereditary'. Tumors exhibiting MLH1 promoter methylation and/or the BRAF V600E mutation were classified as 'sporadic'.

Microdissection and DNA isolation
Three to six tissue sections (2 mm, formalin fixed and paraffinembedded) were deparaffinized and stained with hematoxylin and eosin according to standard protocols. Tumor areas were manually microdissected from HE-stained sections, and genomic tumor DNA was isolated using the Qiagen DNeasy Tissue Kit (Qiagen, Cat. No.: 69506) according to the manufacturer's instructions.

Immunohistochemical staining
Immunohistochemical staining was performed on 2 mm paraffin sections. After deparaffinization in xylol and rehydration in a decreasing series of ethanol baths, the slides were boiled 3 £ 5 minutes in 10 mM citrate buffer (pH 6.0) for antigen retrieval. After boiling, the slides cooled down for 20 minutes, followed by blocking the endogenous peroxidase activity with 0.06% H 2 O 2 (v/v in methanol) for 20 minutes. Subsequently the slides were incubated with 10% horse serum (Vector Laboratories, Cat. No.: S-2000) v/ v in phosphate-buffered saline for 30 min at room temperature to prevent nonspecific antibody binding.

Determination of B2M and RFX5/CIITA mutation status
Mutation analysis of the B2M gene was performed for all tumor specimens by using Sanger sequencing as described previously. 13 For all samples categorized as lack of or barely detectable HLA class II antigen expression, Sanger sequencing of the RFX5 and CIITA genes was performed as previously described 17  Pictures were scanned using a Hamamatsu NanoZoomer Digital Pathology system (Hamamatsu,Hamamatsu City, Japan). A subset of immunohistochemically stained tissue sections (20 tumor regions) was examined by a second observer (M.K.) with respect to CD3-positive T cells. With a Spearman's rank correlation coefficient of 0.96 (one-sided 95%-CI: 0.87-1), the results of the manual counting can be considered reliable.

Statistical analysis
Wilcoxon's rank sum test was applied to compare the distribution of immune cell infiltration depending on B2M mutation status and type of MSI (hereditary or sporadic). With respect to HLA class II antigen status the Jonckheere-Terpstra test was used. Fisher's exact test was applied to compare distributions of categorical data.
For the multivariate analysis with respect to the B2M mutation status, a logistic regression model was set up including age (continuous), gender, MSI type (sporadic/hereditary), CD3positive T cells per 0.25 mm 2 , PDCD1 (PD-1)-positive T cells per 0.25 mm 2 , HLA class II antigen expression and BRAF mutation status as covariates. For the HLA class II antigen expression, an ordered logistic model was applied to the categorized version of the HLA class II antigen expression. Other than e1390640-6 replacing the HLA class II antigen expression by the B2M mutation status, the same covariates were considered. Due to a low number of cases (53 cases for B2M mutation status; 37 wild type vs. 15 mutant; 49 cases for HLA class II antigen; 14 level 0 vs. 10 level 1 vs. 25 level 2), a backward selection was added to the multivariate analysis. The threshold for the selection was chosen at p D 0.5 according to Steyerberg et al. 58 as a tradeoff between selection bias and overestimation bias.
All analyses were performed on the basis of the mean of the immune cell counts over all tumor regions where measurements have been taken. P values smaller than 0.05 were considered statistically significant. The statistical analysis was performed within the statistical software environment R. 59

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.