Whole-cell and single-channel analysis of P-type calcium currents in cerebellar Purkinje cells of leaner mutant mice

Abstract

The leaner (tgla) mutation in mice results in severe ataxia and an overt neurodegeneration of the cerebellum. Positional cloning has revealed that the tgla mutation occurs in a gene encoding the voltage-activated calcium channel alpha1A subunit. The alpha1A subunit is highly expressed in the cerebellum and is thought to be the pore-forming subunit of P- and Q-type calcium channels. In this study we used both whole-cell and single-channel patch-clamp recordings to examine the functional consequences of the tgla mutation on P-type calcium currents. High-voltage-activated (HVA) calcium currents were recorded from acutely dissociated cerebellar Purkinje cells of homozygous leaner (tgla/tgla) and age-matched wild-type (+/+) mice. In whole cell recordings, we observed a marked reduction of peak current density in tgla/tgla Purkinje cells (-35.0 +/- 1.8 pA/pF) relative to that in +/+ (-103.1 +/- 5.9 pA/pF). The reduced whole-cell current in tgla/tgla cells was accompanied by little to no alteration in the voltage dependence of channel gating. In both genotypes, HVA currents were predominantly of the omega-agatoxin-IVA-sensitive P-type. Cell-attached patch-clamp recordings revealed no differences in single-channel conductance between the two genotypes and confirmed the presence of three distinct conductance levels (9, 13-14, and 17-18 pS) in cerebellar Purkinje cells. Analysis of patch open-probability (NPo) revealed a threefold reduction in the open-probability of channels in tgla/tgla patches (0.04 +/- 0.01) relative to that in +/+ (0.13 +/- 0.02), which may account for the reduced whole-cell current in tgla/tgla Purkinje cells. These results suggest that the tgla mutation can alter native P-type calcium channels at the single-channel level and that these alterations may contribute to the neuropathology of the leaner phenotype.

Fig. 1.

HVA calcium currents recorded from wild-type (+/+) and leaner (tg^la/tg^la) Purkinje cells. A, Currents were elicited by a depolarizing test pulse to −10 mV (40 msec) from V_h = −60 mV. Voltage protocol is shown at top. B, Peak currents from +/+ and tg^la/tg^laPurkinje cells were divided by cell capacitance and reported as current densities (pA/pF). Peak current densities for individual cells were plotted versus days of age. Inset, Box plot summary for current densities in +/+ (shaded) and tg^la/tg^la (open) Purkinje cells is shown. Box boundaries represent 25th and 75th percentiles. The horizontal line within eachbox represents the median percentile, andextended lines represent 10th and 90th percentiles. Thefilled square within the box represents the mean, and asterisks and filled diamonds (below and above extended lines) represent 1st and 99th percentiles, respectively. There is a statistically significant difference (p< 0.001) between the means for +/+ (−103.1 ± 5.9 pA/pF;n = 45 cells) and tg^la/tg^la (−35.0 ± 1.8 pA/pF; n = 46 cells) Purkinje cells.

Fig. 2.

Current–voltage relationships in +/+ and tg^la/tg^la Purkinje cells.A, Representative current traces are shown for +/+ and tg^la/tg^laPurkinje cells. Currents were elicited by voltage steps from −80 to +30 mV (200 msec) delivered at 8 sec intervals with V_h = −60 mV. Voltage protocol is shown at top.B, Currents were divided by cell capacitance and reported as current densities (pA/pF). Current density ± SE was plotted versus test voltage. There are statistically significant differences (p < 0.002) in current densities between +/+ (n = 15 cells, 10 mice, 8 litters) and tg^la/tg^la(n = 16 cells, 8 mice, 7 litters) Purkinje cells from −30 to +30 mV. Inset, The scaled activation curve for tg^la/tg^la cells is superimposed on the +/+ activation curve. C, Representative current traces are shown for +/+ and tg^la/tg^la Purkinje cells. Currents were elicited by voltage ramps from −80 to +30 mV (400 msec) with V_h = −60 mV. Voltage protocol is shown attop. D, Currents were divided by cell capacitance and reported as current densities (pA/pF). Current densities were averaged and plotted versus membrane voltage. SEs are shown at peak current densities.

Fig. 3.

Voltage-dependent inactivation in +/+ and tg^la/tg^la Purkinje cells.A, Inactivation records show 24 superimposedtraces of currents elicited by a depolarizing test pulse to −10 mV (120 msec). Before the test pulse, a 2 sec conditioning pulse varying in 5 mV increments between −110 and +5 mV was given.B, Voltage-dependent inactivation curves in +/+ and tg^la/tg^la Purkinje cells are shown. Currents were measured near the end of the pulse and were normalized to the maximum current in each record. Normalized currents ± SE were plotted versus conditioning voltage and fit with a Boltzmann distribution. Inset, Normalized activation curves are superimposed for +/+ and tg^la/tg^la Purkinje cells.

Fig. 4.

Relief of steady-state inactivation in +/+ and tg^la/tg^la Purkinje cells. Currents were elicited by a depolarizing test pulse to −10 mV from the indicated V_h. Currents were divided by cell capacitance and reported as current density. A, Time course of relief of steady-state inactivation after changing V_h from −60 to −80 mV is shown for a +/+ Purkinje cell. B, Time course of relief of steady-state inactivation after changing V_hfrom −60 to −80 mV is shown for a tg^la/tg^la Purkinje cell.Insets, Superimposed current traceselicited from holding potentials of −60 and −80 mV are shown.

Fig. 5.

Antagonist block of HVA current in +/+ and tg^la/tg^la Purkinje cells. Currents were elicited by depolarizing test pulses to −10 mV (40 msec) from V_h = −60 mV at 12 sec intervals. A, Time course of block by Aga-IVA is shown for a +/+ Purkinje cell.Inset, Superimposed current records showtraces before and after application of Aga-IVA (100 nm). B, Time course of block by Aga-IVA is shown for a tg^la/tg^la Purkinje cell. Inset, Superimposed current records showtraces before and after application of Aga-IVA (100 nm). C, Current fractions in +/+ (shaded) and tg^la/tg^la (open) Purkinje cells are shown. Bar graphs with error bars show current remaining after maximum blockade. Percent blockade was corrected for gradual current rundown. The effect of CTX-GVIA was indistinguishable from current rundown in both +/+ (n = 7 cells) and tg^la/tg^la (n= 9 cells) Purkinje cells (nominally 5%). There were no significant differences in the amount of current blockade by nifedipine in +/+ (n = 6 cells) and tg^la/tg^la (n= 9 cells) cells or by Aga-IVA in +/+ (n = 4) and tg^la/tg^la (n= 5 cells) cells.

Fig. 6.

Conductance of single calcium channels in +/+ and tg^la/tg^la Purkinje cells. Single-channel currents were recorded from cell-attached patches with 110 mm Ba²⁺ in the patch pipette. Currents were elicited by 300 msec steps to the indicated potential from a holding potential of −60 mV at 4 sec intervals. Currents were low-pass filtered at 2 kHz and digitally acquired at 10 kHz.A, Single-channel current records from a +/+ patch are shown. Three open levels are indicated on the bottom trace. B, Single-channelI–V plot yields three distinct slope conductances as labeled. C, Single-channel current records from a tg^la/tg^la patch are shown. Open levels are indicated on the bottom trace. D, Single-channelI–V plot yields three distinct slope conductances as labeled.

Fig. 7.

Patch open-probability (NP_o) for cell-attached patches of +/+ and tg^la/tg^la Purkinje cells. Currents were elicited by 300 msec steps to −10 mV from a holding potential of −60 mV at 4 sec intervals. Currents were low-pass filtered at 2 kHz and digitally acquired at 10 kHz. Currents are displayed here with a 1 kHz filter imposed. A, Ten consecutive current traces recorded from a +/+ patch are shown. B, For the +/+ patch in A,NP_o has been calculated for individual sweeps. NP_o is displayed for 45 consecutive sweeps in which the total patchNP_o is 0.10. C, Ten consecutive current traces recorded from a tg^la/tg^la patch are shown.D, For the tg^la/tg^la patch inC, NP_o is displayed for 45 consecutive sweeps in which the total patchNP_o is 0.02.

Fig. 8.

Mean open times of calcium channels from +/+ and tg^la/tg^la Purkinje cells. Calcium currents were elicited by 300 msec steps to −10 mV from a holding potential of −60 mV at 4 sec intervals. Currents were low-pass filtered at 2 kHz and digitally acquired at 10 kHz. A, Single-channel current records from a +/+ patch. B, Open-time histogram for the +/+ patch shown in A, fitted with a single exponential. C, Single-channel current records from a tg^la/tg^la patch.D, Open-time histogram for the tg^la/tg^la patch shown inC, fitted with a single exponential.