The Ca(V)2.3 Ca(2+) channel subunit contributes to R-type Ca(2+) currents in murine hippocampal and neocortical neurones

Abstract

Different subtypes of voltage-dependent Ca(2+) currents in native neurones are essential in coupling action potential firing to Ca(2+) influx. For most of these currents, the underlying Ca(2+) channel subunits have been identified on the basis of pharmacological and biophysical similarities. In contrast, the molecular basis of R-type Ca(2+) currents remains controversial. We have therefore examined the contribution of the Ca(V)2.3 (alpha(1E)) subunits to R-type currents in different types of central neurones using wild-type mice and mice in which the Ca(V)2.3 subunit gene was deleted. In hippocampal CA1 pyramidal cells and dentate granule neurones, as well as neocortical neurones of wild-type mice, Ca(2+) current components resistant to the combined application of omega-conotoxin GVIA and MVIIC, omega-agatoxin IVa and nifedipine (I(Ca,R)) were detected that were composed of a large R-type and a smaller T-type component. In Ca(V)2.3-deficient mice, I(Ca,R) was considerably reduced in CA1 neurones (79 %) and cortical neurones (87 %), with less reduction occurring in dentate granule neurones (47 %). Analysis of tail currents revealed that the reduction of I(Ca,R) is due to a selective reduction of the rapidly deactivating R-type current component in CA1 and cortical neurones. In all cell types, I(Ca,R) was highly sensitive to Ni(2+) (100 microM: 71-86 % block). A selective antagonist of cloned Ca(V)2.3 channels, the spider toxin SNX-482, partially inhibited I(Ca,R) at concentrations up to 300 nM in dentate granule cells and cortical neurones (50 and 57 % block, EC(50) 30 and 47 nM, respectively). I(Ca,R) in CA1 neurones was significantly less sensitive to SNX-482 (27 % block, 300 nM SNX-482). Taken together, our results show clearly that Ca(V)2.3 subunits underlie a significant fraction of I(Ca,R) in different types of central neurones. They also indicate that Ca(V)2.3 subunits may give rise to Ca(2+) currents with differing pharmacological properties in native neurones.

Figure 1. Pharmacological isolation of Ca²⁺ currents resistant to organic Ca²⁺ channel antagonists (I_Ca,R) by combined application of ω-conotoxin (CgTx) GVIA (2 μM), ω-CgTx MVIIC (3 μM), ω-agatoxin (AgaTx) IVa (200 nm) and nifedipine (10 μM) in wild-type mice

A, Ba²⁺ currents were elicited with voltage jumps to 0 mV (200 ms) following a conditioning prepulse to −100 mV (2 s, see inset). The holding potential in this recording was −50 mV. The current trace following the saturation of the block is marked with an asterisk. B, time course of block of the peak Ba²⁺ current by combined application of the Ca²⁺ channel antagonists (horizontal bar). Care was always taken to ensure that the block was saturated before analysis of I_Ca,R current properties.

Figure 2. I_Ca,R is considerably reduced in Ca_V2.3(-/-) mice

A, analysis of Ca_V2.3 protein levels using Western blot analysis of microsomal membrane proteins. Microsomes (24 μg) were isolated from the brains of wild-type mice (lane 1), from heterozygous litter mates (lane 2) and from Ca_V2.3 null mutants (lane 3). Microsomal membranes from untransfected (lane 4; 5 μg) and stably transfected with Ca_V2.3 human embryonic kidney (HEK)-293 cells (lane 5; 2 μg) were used as negative and positive controls, respectively. The primary antibody (Anti-N195B) is directed against an epitope of Ca_V2.3 common to all known Ca_V2.3 splice variants. A polypeptide of 246 kDa (Ca_V2.3) was detected in wild-type and heterozygous mice as well as in stably transfected HEK-293 cells (arrow). No staining was observed at this position in Ca_V2.3 null mutant mice or untransfected HEK-293 cells (lanes 3 and 4). Unspecific staining was detected as a faint band at 205 kDa and a major band (*), neither of which is related to Ca_V2.3 because they were also detected after preabsorption of the serum by the antigenic peptide (not shown). B, I_Ca,R in CA1 pyramidal neurones (CA1), dentate granule cells (DG) and neocortical neurones (Cx) in both wild-type (upper traces) and Ca_V2.3-deficient mice (lower traces). I_Ca,R was elicited with the voltage-step protocol shown in the inset. Calibration bars apply to all traces. Ca, the amplitude of I_Ca,R elicited with the voltage step to −10 mV was significantly (*P < 0.05) decreased in Ca_V2.3-deficient mice (open bars) compared to wild-type mice (filled bars) in all cell types studied. The amplitude of I_Ca,R elicited with command pulses to −10 mV was normalised to cell capacitance. Cb, normalised amplitude of the current component blocked by the combination of Ca²⁺ channel antagonists used to isolate I_Ca,R (see Fig. 1A). No difference between wild-type mice and mice lacking Ca_V2.3 subunits was detected.

Figure 3. Selective reduction of a rapidly deactivating, putative R-type current in Ca_V2.3(-/-) mice

A, deactivation kinetics were determined following a brief (5 ms) test pulse following a conditioning prepulse to −100 mV (see inset). The holding potential for these recordings was −80 mV. Recordings shown on a semilogarithmic scale were generated by subtracting traces obtained after complete blockade of inward currents with 1 mm Ni²⁺. The resulting Ba²⁺ current tails were fitted with a biexponential function shown as a dashed line superimposed on representative example traces. Traces are shown beginning from the peak amplitude. Averaged time constants are given in Table 1. B, the amplitude contributions of the fast- (Ba) and the slowly deactivating components (Bb) were derived from the fits, normalised to the cell capacitance and averaged in wild-type (filled bars) and Ca_V2.3-deficient mice (open bars). The rapidly decaying, putative R-type component was considerably reduced in Ca_V2.3-deficient mice (*P < 0.05).

Figure 4. Voltage dependence of I_Ca,R in wild-type and Ca_V2.3-deficient mice

A, voltage dependence of activation (Aa) and inactivation (Ab) in a CA1 neurone from a wild-type mouse. The activation behaviour was analysed with voltage jumps to various command voltages (200 ms) following a conditioning prepulse to −100 mV (2 s, see inset). Inactivation was induced with a 5 s conditioning pulse to various voltages followed by a test pulse to −10 mV (50 ms, see inset). B, voltage dependence of activation (Ba) and inactivation (Bb) in a CA1 neurone from a Ca_V2.3-deficient mouse. C, D, E, voltage-dependent activation and inactivation behaviour for wild-type mice (filled symbols) and Ca_V2.3-deficient mice (open symbols) in CA1 neurones (C), dentate granule neurones (DG, D) and cortical neurones (Cx, E). Data points for the steady-state activation curve were obtained by calculating the conductance from peak Ba²⁺ current values (see Aa, Ba), and plotting normalised and averaged values vs. the command voltage. The inactivation curve was constructed from normalised and averaged peak conductances and plotted vs. the voltage of the conditioning pulse. Boltzmann functions were fitted to the data points using the Levenberg-Marquardt least-squares algorithm and are shown superimposed on the data points. The half-maximal activation and inactivation voltages averaged across individual experiments are given in Table 1.

Figure 5. Pharmacological properties of I_Ca,R

A, time course of a representative experiment during application of 10–300 nm SNX-482 (Aa, see bars for duration of application). The amplitude of I_Ca,R in a cortical neurone from a wild-type mouse is depicted over time. Current traces elicited at the time points indicated by the lower-case letters are shown in panel Ab. The voltage step is shown in the inset and was applied following a 2 s hyperpolarising prepulse to −100 mV. The holding potential in all pharmacological experiments was −50 mV. B, concentration dependence of I_Ca,R block by SNX-482 in CA1 neurones (squares), dentate granule cells (circles) and cortical neurones (triangles) in wild-type mice. Data points were fitted with the logarithmically transformed Hill equation (eqn (4), see Methods) and the fitted curves are superimposed on the data points (dashed curve: cortex). The fitted curve for CA1 neurones is given for illustrative purposes only. Note that block by SNX-482 does not seem to saturate completely during some applications, either due to a slow onset of the block, or to some degree of unavoidable rundown. C, fraction of Ba²⁺ current blocked by application of 100 μM Ni²⁺ in the different neurone types from wild-type mice.