Compensatory regulation of Cav2.1 Ca2+ channels in cerebellar Purkinje neurons lacking parvalbumin and calbindin D-28k

Abstract

Ca(v)2.1 channels regulate Ca(2+) signaling and excitability of cerebellar Purkinje neurons. These channels undergo a dual feedback regulation by incoming Ca(2+) ions, Ca(2+)-dependent facilitation and inactivation. Endogenous Ca(2+)-buffering proteins, such as parvalbumin (PV) and calbindin D-28k (CB), are highly expressed in Purkinje neurons and therefore may influence Ca(v)2.1 regulation by Ca(2+). To test this, we compared Ca(v)2.1 properties in dissociated Purkinje neurons from wild-type (WT) mice and those lacking both PV and CB (PV/CB(-/-)). Unexpectedly, P-type currents in WT and PV/CB(-/-) neurons differed in a way that was inconsistent with a role of PV and CB in acute modulation of Ca(2+) feedback to Ca(v)2.1. Ca(v)2.1 currents in PV/CB(-/-) neurons exhibited increased voltage-dependent inactivation, which could be traced to decreased expression of the auxiliary Ca(v)beta(2a) subunit compared with WT neurons. Although Ca(v)2.1 channels are required for normal pacemaking of Purkinje neurons, spontaneous action potentials were not different in WT and PV/CB(-/-) neurons. Increased inactivation due to molecular switching of Ca(v)2.1 beta-subunits may preserve normal activity-dependent Ca(2+) signals in the absence of Ca(2+)-buffering proteins in PV/CB(-/-) Purkinje neurons.

Fig. 1.

Ca²⁺-dependent inactivation of P-type currents in mouse cerebellar Purkinje neurons. A: current–voltage (I–V) relationships for a Ca²⁺ current (I_Ca) evoked by 50-ms steps from −60 mV to various voltages. Shown are representative current traces and voltage protocol (top) and I–V relationship (bottom) in cells before (control, filled circles) and after (+Aga-IVA, open circles) extracellular perfusion with ω-agatoxin-IVA (500 nM). Points represent means ± SE (n = 10). B: inactivation of P-type currents in Purkinje neurons and Ca_v2.1 (α₁2.1, β_2a, α₂δ) in transfected HEK293T cells. In Purkinje neurons, I_Ca and Ba²⁺ current (I_Ba) were evoked by 2-s pulses from −60 to −10 or −20 mV, respectively, and were evoked in transfected HEK293T cells by 2-s pulses from −80 to +10 or 0 mV, respectively. The −10-mV difference in test pulse used for I_Ba accounts for the shift in activation voltage when using Ba²⁺ as the charge carrier. Voltage protocol and current traces for I_Ca (black) and I_Ba (gray) are shown (top). I_res/I_pk (current amplitude at end of 2-s pulse normalized to peak current amplitude) is plotted below for Purkinje neurons (left) and transfected HEK293T cells (right). Parentheses indicate numbers of cells. *P < 0.01 by t-test. C: Ca²⁺-dependent facilitation during repetitive depolarizations in Ca_v2.1-transfected HEK293T cells. I_Ca and I_Ba were recorded during 5-ms steps from −80 to +10 mV (I_Ca) or 0 mV (I_Ba) delivered at 100 Hz. Fractional current represents test current amplitude normalized to the first in the train and is plotted against time. Every third data point is plotted. D: same as in C except that pulses were from −60 to 0 mV (I_Ca) or −10 mV (I_Ba) and applied to Purkinje neurons. In C and D, representative I_Ca and voltage protocols are shown above averaged data. Dashed line represents initial current amplitude.

Fig. 2.

Ca²⁺-dependent facilitation in Purkinje neurons during trains of action potential (AP) waveforms. A: spontaneous APs recorded in current-clamp (left) were used to construct an average AP waveform (right) with amplitude (+27 mV) and half-width (0.75 ms) indicated. B: representative I_Ca and I_Ba evoked by the AP waveform stimulus in A delivered at 200 Hz. Dashed line indicates current amplitude of the first pulse. C: increased Ca²⁺-dependent facilitation with higher frequency stimulation. Current responses to AP waveforms were measured at 50, 100, and 200 Hz. Fractional current represents test current amplitude normalized to the first in the train and plotted against time. For 100- and 200-Hz data, every third and fourth data point is plotted, respectively.

Fig. 3.

Ca_vβ subunits influence the profile of Ca_v2.1 currents during AP stimuli. I_Ca and I_Ba were evoked by AP waveforms at 200 Hz. Representative I_Ca (top) and fractional current measured as in Fig. 2C (bottom) in Purkinje neurons (A) and HEK293T cells transfected with α₁2.1, α₂δ, and β_2a (B) or β₄ (C). Parentheses indicate numbers of cells. Results in A–C and for cells cotransfected with β_2a and β₄ for I_Ca (D) and I_Ba (E) are superimposed on the same graph for comparison. For clarity, error bars were omitted in D and E and, for each graph, every third data point is plotted. For HEK293T cells cotransfected with both β_2a and β₄, results from exemplar cells are shown.

Fig. 4.

Characterization of P-type currents in PV/CB^−/− Purkinje neurons. A: representative current I_Ca (A) and I–V relationship (B) in cells before (filled circles) and after (open circles) exposure to ω-agatoxin IVA (500 nM). Points represent means ± SE (n = 12). CB, calbindin D-28k; PV, parvalbumin.

Fig. 5.

Increased voltage- but not Ca²⁺-dependent inactivation (CDI) of Ca_v2.1 currents in PV/CB^−/− Purkinje neurons. A: I_Ca and I_Ba were evoked by 2-s pulses from −60 to 0 mV (I_Ca) or −10 mV (I_Ba) in wild-type (WT) and PV/CB^−/− neurons. CDI represents the difference in I_res/I_pk for I_Ca and I_Ba in WT and PV/CB^−/− neurons. B: comparison of I_res/I_pk for I_Ca and I_Ba (measured with 2 mM extracellular Ba²⁺) in WT (black traces) and PV/CB^−/− neurons (gray traces). *P < 0.05 by t-test. Parentheses indicate numbers of cells.

Fig. 6.

Increased inactivation of P-type currents during high-frequency stimulation in PV/CB^−/− Purkinje neurons. I_Ca was evoked by AP waveform stimuli given at 50 (A), 100 (B), or 200 Hz (C) in WT (filled circles) and PV/CB^−/− (open circles) Purkinje neurons. Every other point is plotted for 200-Hz data. D: normalized I_Ca represents the grand average of current amplitude for the last 10 pulses (±SE) plotted against stimulation frequency for WT and PV/CB^−/− neurons. **P < 0.01; *P < 0.05 compared with WT by t-test.

Fig. 7.

Altered Ca_v2.1 subunit expression in PV/CB^−/− Purkinje neurons. A: control experiment in which cerebellar tissue or pools of 5–10 Purkinje neurons from WT or PV/CB^−/− mice were subjected to polymerase chain reaction (PCR) with primers specific for calbindin D-28k (Calb1, CB), calretinin (Calb2, CR), or GAPDH. B: representative experiment showing amplification of all 4 β subunits in WT Purkinje neurons (n = 5) and the absence of β₂ in PV/CB^−/− neurons (n = 5). C: PCR analysis of Ca_vβ subunits (β₁–β₄) in cerebellar tissue from WT and PV/CB^−/− mice. D: average results indicating percentage of individual neurons in WT and PV/CB^−/− mice in which β₂ was detected (left). Parentheses indicate numbers of cells. *P < 0.05 by t-test. Right: results from quantitative PCR from 5 to 10 Purkinje neurons using β_2a-specific primers. Relative levels of β_2a in WT and PV^−/− neurons were determined as described in methods. Shown are averaged results from 5 independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 8.

No differences in spontaneous APs in WT and PV/CB^−/− Purkinje neurons. Spontaneous APs were recorded from (A) WT and (B) PV/CB^−/− neurons in current clamp without injected current. C: average AP waveform for WT (black trace, n = 11) and PV/CB^−/− (gray trace, n = 10) neurons. For each neuron, averages are from all APs recorded in 10 consecutive sweeps of 500-ms duration. D–G: parameters for spontaneous APs in WT (circles) and PV/CB^−/− (squares) neurons. Afterhyperpolarization (AHP) amplitude (D) was determined as the difference between the membrane potential measured 5 ms before the AP peak (double arrows in C) and the minimum membrane potential after the AP peak (single arrow in C). AP half-width (E) represents the duration of the AP at half-maximal amplitude from the threshold membrane potential. AP threshold (F) was measured at 5–15% of the tangential slope of the threshold depolarization. Mean interspike interval (ISI, G) is the average duration between peaks of the APs. Each symbol represents averaged data recorded as in A from one Purkinje neuron. Population averages are indicated with filled symbol (±SE).