Maturation of rat cerebellar Purkinje cells reveals an atypical Ca2+ channel current that is inhibited by omega-agatoxin IVA and the dihydropyridine (-)-(S)-Bay K8644

Abstract

To determine if the properties of Ca2+ channels in cerebellar Purkinje cells change during postnatal development, we recorded Ca2+ channel currents from Purkinje cells in cerebellar slices of mature (postnatal days (P) 40-50) and immature (P13-20) rats. We found that at P40-50, the somatic Ca2+ channel current was inhibited by omega-agatoxin IVA at concentrations selective for P-type Ca2+ channels (approximately 85%; IC50, <1 nM) and by the dihydropyridine (-)-(S)-Bay K8644 (approximately 70%; IC50, approximately 40 nM). (-)-(S)-Bay K8644 is known to activate L-type Ca2+ channels, but the decrease in current was not secondary to the activation of L-type channels because inhibition by (-)-(S)-Bay K8644 persisted in the presence of the L-type channel blocker (R,S)-nimodipine. By contrast, at P13-20, the current was inhibited by omega-agatoxin IVA (approximately 86%; IC50, approximately 1 nM) and a minor component was inhibited by (R,S)-nimodipine (approximately 8%). The dihydropyridine (-)-(S)-Bay K8644 had no clear effect when applied alone, but in the presence of (R,S)-nimodipine it reduced the current (approximately 40%), suggesting that activation of L-type channels by (-)-(S)-Bay K8644 masks its inhibition of non-L-type channels. Our findings indicate that Purkinje neurons express a previously unrecognized type of Ca2+ channel that is inhibited by omega-agatoxin IVA, like prototypical P-type channels, and by (-)-(S)-Bay K8644, unlike classical P-type or L-type channels. During maturation, there is a decrease in the size of the L-type current and an increase in the size of the atypical Ca2+ channel current. These changes may contribute to the maturation of the electrical properties of Purkinje cells.

Figure 4

Whole-cell Ca²⁺ channel currents in mature cerebellar Purkinje cells run down slowly and are almost completely blocked by P-type-selective concentrations of ω-agatoxin IVA A, example of a current evoked by a depolarizing voltage ramp (−96 to +74 mV) applied to a Purkinje cell in the whole-cell configuration, approximately 16 min after establishing the whole-cell recording configuration and about 14 min after commencement of superfusion of the cell with an extracellular solution containing 2 mm Ba²⁺ as the main divalent cation, and 1 μm TTX to block Na⁺ channels. The horizontal dashed line (left) denotes the zero current level. A linear leak current was estimated by fitting a straight line (dotted line) to the current recorded during the negative potentials of the voltage ramp that did not evoke an inward current (−96 to ∼−65 mV). It was extrapolated over the whole length of the recording, and the peak inward Ca²⁺ channel current, I_pk, was measured from the dotted line. B, scatter diagram and box plot of whole-cell I_pk measurements in many mature Purkinje cells, made about 10 min after establishing each whole-cell recording. C, plots of whole-cell I_pk, normalized by size at time zero, against time in 13 cells (grey circles) showing that the size of the current runs down slowly (∼6% decrease in 10 min, measured from the mean I_pk, empty circles). D, time course of block and unblock of the inward current recorded in a single cell by 100 μm Cd²⁺. The superimposed traces to the right show the effect of Cd²⁺ on the currents. Note that the decrease in inward current was not accompanied by a change in holding current (horizontal dashed line indicates zero current level for all three traces). E, comparison of the time course of block of whole-cell I_pk by superfusion of 100 nmω-agatoxin IVA on to five cells (filled circles, mean; vertical bars, ± s.e.m.), with a plot of the mean whole-cell I_pk recorded in 13 cells in the absence of drug (open circles, as in C). The superimposed current traces to the right show the effect of 100 nmω-agatoxin IVA on currents evoked by depolarizing voltage ramps in a single cell. The inhibition of the inward current by ω-agatoxin IVA was not accompanied by a change in holding current (horizontal dashed line denotes zero current level for all three traces). It revealed an outward current (trace above the dotted line), as did the inhibition by Cd²⁺ (see D and Methods).

Figure 1

Cell-attached recording of Ca²⁺ channel currents carried by 5 mm Ba²⁺ in the soma of Purkinje cells in cerebellar slices A, superimposed current traces (grey) recorded from a single patch (P43 animal), showing constancy in the shape of currents evoked by 30 voltage ramps (top) applied at 0.2 Hz. The average of these currents is shown by the black trace. A linear leak current (dashed line) was estimated by fitting a straight line to the linear part of the mean trace (a–b) and extrapolating it over the full range of the ramp. B, the mean Ca²⁺ channel current obtained by subtracting the leak current from the mean total current. The size of the Ca²⁺ channel current was measured as I_pk from the zero current level (dotted line) as indicated. C, example of an unstable cell-attached recording. The current traces were evoked by the 1st, 6th, 12th, 18th and 24th voltage ramp, and illustrate a characteristic leftward shift accompanied by a decrease in size.

Figure 2

Pharmacological confirmation that inward currents evoked by depolarizing voltage ramps in somatic patches of mature cerebellar Purkinje cells (P40–50) are mediated mostly by P-type Ca²⁺ channels A, superimposed Ca²⁺ channel currents recorded with (bottom, 13 patches) and without 100 μm Cd²⁺ (top, 20 patches) in the patch-pipette, from cells within the same slices. In this and subsequent figures, each current trace is the mean of currents evoked by ∼15–35 voltage ramps in a single patch, from which a leak current has been subtracted, as in Fig. 1. B, concentration dependence of inhibition of I_pk by Cd²⁺. Symbols and vertical bars plot means ± s.e.m. of measurements of I_pk in the numbers of patches given in parentheses. The continuous curve is the best fit with the logistic equation, for which IC₅₀ is 40 μm, Hill slope is 1.6, max − min is 28.6 pA (100%) (max is the maximum I_pk in the absence of drug, and min is the minimum current remaining). C, superimposed Ca²⁺ channel currents recorded in 51 patches with control pipette solution, and in 41 patches with 30 nmω-agatoxin-IVA in the pipette, from the same slices. D, concentration dependence of the decrease of I_pk by ω-agatoxin-IVA. The continuous line is the best fit of the logistic equation, with IC₅₀ 0.4 nm, Hill slope 0.59, max − min 26 pA (85%). E, superimposed Ca²⁺ channel currents recorded with drug-free pipette solution (eight patches) and with 10 μmω-conotoxin MVIIC in the pipette (six patches) showing 99% inhibition. F, concentration dependence of the rate of Ca²⁺ channel inhibition by ω-conotoxin MVIIC. Plots illustrate the time course of inhibition by three different concentrations in three different patches. Each is fitted with a single exponential function (continuous line), characterized by the time constants shown. The zero time point of each exponential was defined as the time of seal formation. Recordings started ∼60 s (1 μm) or ∼80 s (100 nm, 10 μm) after seal formation.

Figure 3

Ca²⁺ channel currents in somatic patches of mature cerebellar Purkinje cells are reduced by (−)-(S)-Bay K8644 This effect does not involve the activation of L-type channels. A, Ca²⁺ channel currents evoked in cell-attached patches by depolarizing voltage ramps in the absence (40 patches) and presence of 300 nm (−)-(S)-Bay K8644 (27 patches) in the patch-pipette, recorded from the same slices. B, concentration dependence of the decrease of I_pk by (−)-(S)-Bay K8644. Symbols and vertical bars plot means ± s.e.m. Numbers of patches for each concentration are given in parentheses. The continuous line is the best fit of the logistic equation, for which IC₅₀ is 40 nm, Hill slope is 2.9, max − min is 21.1 pA (70%). C, D and E, bar charts showing that mean I_pk in cell-attached patches is not altered by the inclusion of 1 μm (R,S)-nimodipine (P = 0.55, Student's t test) or 30 nm or 3 μm FPL 64176 (P = 0.24, ANOVA) in the pipette, whereas 300 nm (−)-(S)-Bay K8644 reduced I_pk by 64% when applied in the presence of 1 μm (R,S)-nimodipine (*P = 0.011, Student's t test). F, superimposed mean (black line) ± s.e.m. (grey) of currents evoked in multiple patches (numbers in parentheses) by voltage jumps, demonstrating inhibition of the currents by (−)-(S)-Bay K8644 in the presence or absence of (R,S)-nimodipine. G, the mean currents in F normalized to the maximum current, showing that only a minor change in the activation kinetics (arrow) accompanies the inhibition of the currents. H, plot of means ± s.e.m. of I_pk against different concentrations of (+)-(R)-Bay K8644. I, bar chart illustrating reduction in I_pk by racemic Bay K8644 (*P = 0.003, Student's t test).

Figure 5

Whole-cell Ca²⁺ channel currents in mature Purkinje cells are inhibited by local application of ω-agatoxin IVA or (S)-Bay K8644 to the soma from a micropipette A, digitized image illustrating the application of drug from a micropipette (right) to the soma of a Purkinje cell, that had been previously cleaned of overlying tissue by applying a stream of extracellular solution from a different pipette (not in view), and to which a recording-pipette (left) is attached in a whole-cell configuration. B, plot of whole-cell I_pk normalized to I_pk at time zero (circles and vertical bars: means ± s.e.m.), against time for recordings in which a micropipette (pipette symbol) was used to apply drug-free extracellular solution to the soma (open circles, eight cells) or solution containing 300 nmω-agatoxin IVA (filled circles, six cells). Time zero denotes the start of the application. In this and subsequent figures, the concentration in the micropipette is denoted by the subscript ‘pip’, and diagonal lines to the right of the pipette symbol signify maintained ejection of solution from the micropipette. The current traces in the inset show the inhibition of the inward current by ω-agatoxin IVA in a single cell. C, plots of normalized whole-cell I_pk (mean ± s.e.m.) against time showing that, when compared with currents recorded in drug-free solution (grey circles, same data as in B) (R,S)-nimodipine did not reduce I_pk(1 μm_pip, red circles, seven cells; 10 μm_pip, open circles, three cells), whereas (−)-(S)-Bay K8644 inhibited the peak current (3 μm_pip, open triangles, nine cells) and this inhibition was not diminished by inclusion of 1 μm (R,S)-nimodipine in the micropipette (green triangles, five cells). D, plots of whole-cell I_pk(mean ± s.e.m.) against time for recordings in which the cell was superfused for 7–10 min with DMSO (0.04%) or (R,S)-nimodipine 1 or 10 μm, before the local application of DMSO (open circles, seven cells) or (R,S)-nimodipine (purple circles, nine cells) or a mixture of (−)-(S)-Bay K8644 plus (R,S)-nimodipine at two concentrations (green triangles, four cells; open triangles, three cells). The concentration of DMSO is equivalent to that in the solution containing 3 μm (−)-(S)-Bay K8644 and 1 μm (R,S)-nimodipine. E, time courses of whole-cell I_pk in two experiments showing reversibility of inhibition by (−)-(S)-Bay K8644. Each cell was superfused with (R,S)-nimodipine 1 or 10 μm, the perfusion was stopped (−)-(S)-Bay K8644 (3 or 30 μm_pip) in the presence of (R,S)-nimodipine 1 or 10 μm_pip was applied to the soma for 15 min, the micropipette was removed and superfusion of the cell by (R,S)-nimodipine was restarted. F, current traces showing the effect of (−)-(S)-Bay K8644 on currents recorded in a single cell, in the presence of (R,S)-nimodipine.

Figure 6

Ca²⁺ channel currents in somatic patches of young cerebellar Purkinje cells (P13–20) are inhibited by ω-agatoxin IVA but not by (−)-(S)-Bay K8644 A, concentration dependence of inhibition of inward currents (5 mm Ba²⁺) evoked by depolarizing voltage ramps in cell-attached patches in the soma by ω-agatoxin IVA included in the recording pipette. Symbols and vertical bars plot mean ± s.e.m. of I_pk measured in the numbers of patches given in parentheses. The continuous curve is the best fit of the logistic equation to points at 0–100 nm, for which IC₅₀ is 1.3 nm, Hill slope is 0.6, max − min is 35.3 pA (86%). The dotted line indicates that the points at 1 and 3 μm, which are concentrations not selective for P-type channels, were excluded from the fit. B, mean Ca²⁺ channel currents (black) ± s.e.m. (grey) generated by averaging leak-subtracted currents evoked by depolarizing voltage ramps in 11 somatic cell-attached patches in the absence of drug, and in 14 patches in the presence of 300 nm (−)-(S)-Bay K8644. C, average currents (black) ± s.e.m. (grey) evoked by voltage jumps in cell-attached patches in the absence (nine cells) and presence of 300 nm (−)-(S)-Bay K8644 in the pipette (five cells).

Figure 7

Whole-cell Ca²⁺ channel currents in immature Purkinje cells are inhibited by ω-agatoxin IVA, or by (−)-(S)-Bay K8644 when applied in the presence of (R,S)-nimodipine A, example of a whole-cell current evoked by a depolarizing voltage ramp (−97 to +73 mV) applied to a P16 Purkinje cell in the whole-cell configuration, superfused with an extracellular solution containing 2 mm Ba²⁺ and 1 μm TTX. The horizontal dashed line (left) denotes the zero current level. The dotted line denotes the estimated linear leak current, from which inward current (I_pk) was measured. B, scatter diagram and box plot of whole-cell I_pk measurements in many P13–20 Purkinje cells, 7–10 min after establishing the whole-cell recording configuration. C, plot of whole-cell I_pk(mean ± s.e.m., after normalization to current size at time zero) against time demonstrates slow rundown of the inward current (4% in 10 min, open circles) and inhibition of the current in two cells by the inclusion of 100 nmω-agatoxin IVA in the superfusate (filled circles and squares). D, plot of whole-cell I_pk(mean ± s.e.m.) against time for recordings in which superfusion of the cells by 2 mm Ba²⁺ solution was stopped and 2 mm Ba²⁺ solution containing various drugs was applied to the soma from a micropipette (pipette symbol and diagonal lines). When the micropipette solution was drug free (grey circles, five cells) or contained 0.03% DMSO (open circles, four cells, same concentration as in 3 μm (−)-(S)-Bay K8644), the currents decreased in size by ∼6% in 10 min. When the micropipette contained 3 μm (−)-(S)-Bay K8644 (green triangles, four cells), there appeared to be a small decrease in current size relative to the mean current in DMSO at 10 min but not at 20 min after start of application. When the micropipette contained 300 nmω-agatoxin IVA (open squares, three cells), there was a clear inhibition of the current (∼40%). E, plots of whole-cell I_pk(mean ± s.e.m.) against time for recordings in which the cell was superfused for 7–10 min with DMSO (0.04%, same concentration as in 3 μm (−)-(S)-Bay K8644 + 1 μm (R,S)-nimodipine) or (R,S)-nimodipine 1 or 10 μm, before the local application of DMSO (empty circles, four cells) or (R,S)-nimodipine (1 μm_pip, purple circles, three cells) or a mixture of (−)-(S)-Bay K8644 plus (R,S)-nimodipine at two concentrations (green triangles, five cells; empty triangles, four cells). The currents were inhibited by (R,S)-nimodipine by ∼8%, relative to the mean I_pk in DMSO, and by (−)-(S)-Bay K8644 by ∼42%, relative to the currents in (R,S)-nimodipine. F, time courses of whole-cell I_pk in two experiments showing reversibility of inhibition by (−)-(S)-Bay K8644. Each cell was superfused with (R,S)-nimodipine 1 or 10 μm, the perfusion was stopped (−)-(S)-Bay K8644 (3 or 30 μm_pip) in the presence of (R,S)-nimodipine (1 or 10 μm_pip) was applied to the soma from a micropipette for 13 min, the pipette was removed and superfusion of the cell by (R,S)-nimodipine was restarted. G, current traces showing the effect of (−)-(S)-Bay K8644 on currents recorded in a single cell, in the presence of (R,S)-nimodipine.