Decoding of synaptic voltage waveforms by specific classes of recombinant high-threshold Ca(2+) channels

Abstract

Studies suggest that the preferential role of L-type voltage-sensitive Ca(2+) channels (VSCCs) in coupling strong synaptic stimulation to transcription is due to their selective activation of local chemical events. However, it is possible that selective activation of the L-type channel by specific voltage waveforms also makes a contribution. To address this issue we have examined the response of specific Ca(2+) channel types to simulated complex voltage waveforms resembling those encountered during synaptic plasticity (gamma and theta firing frequency). L-, P/Q- and N-type VSCCs (alpha1C, alpha1A, alpha1B/beta1B/alpha2delta, respectively) were all similarly activated by brief action potential (AP) waveforms or sustained step depolarization. When complex waveforms containing large excitatory postsynaptic potentials (EPSPs), APs and spike accommodation were applied under voltage clamp we found that the integrated L-type VSCC current was approximately three times larger than that produced by the P/Q- or N-type Ca(2+) channels (gamma frequency 1 s stimulation). For P/Q- or N-type channels the complex waveforms led to a smaller current than that expected from the response to a simple 1 s step depolarization to 0 or +20 mV. EPSPs present in the waveforms favoured the inactivation of P/Q- and N-type channels. In contrast, activation of the L-type channel was dependent on both EPSP- and AP-mediated depolarization. Expression of P/Q-type channels with reduced voltage-dependent inactivation (alpha1A/beta2A/alpha2delta) or the use of hyperpolarized intervals between AP stimuli greatly increased their response to complex voltage stimuli. We propose that in response to complex synaptic voltage waveforms P/Q- and N-type channels can undergo selective voltage-dependent inactivation leading to a Ca(2+) current mediated predominantly by L-type channels.

Figure 2. Ca²⁺ currents in response to simulated waveforms

A, the upper panel shows 1 s duration computer-simulated voltage waveforms used as command potentials to activate the VSCCs (delivered from −70 mV). The lower three panels show the Ca²⁺ current at 37 °C in response to the corresponding voltage waveform above them. B, group data for the results shown in A. Each column was obtained by integrating the total Ca²⁺ current in response to the corresponding voltage waveform (units pC) and subsequently normalizing this to the peak current response (units nA) of a preceding step depolarization (see Methods; expressed as pC nA_peak⁻¹). The results indicate a considerably larger integrated Ca²⁺ current mediated by the L-type channel in response to complex voltage waveforms. L-type channel activation in response to the repetitive, plateau, and strong theta waveforms resulted in a significantly larger normalized Ca²⁺ integrated current than that observed from P/Q- or N-type channels (P < 0.001; n > 10 for all groups). In the case of the weak theta waveform only a modest difference was observed between the L- and P/Q-type channels (P = 0.037), while the N-type channel response was significantly smaller (P < 0.001). Compared to the other two channel types, L-type VSCCs showed a stronger preference for the plateau waveform than the other 3 waveforms given. We have illustrated this difference (comparison between plateau and other waveforms within a channel type) on the figure using asterisks: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 1. Activation and inactivation of VSCCs in response to step depolarization

A, at room temperature (≈22 °C) L-, P/Q- and N-type channels expressed in HEK 293 cells have relatively slow activation and inactivation kinetics (1 s step depolarization from −70 to 0 mV, except +20 mV for N-type). B, at 37 °C all three channel types studied exhibited dramatically faster activation and inactivation. Overplotted normalized records (to peak current) of L-, P/Q- and N-type VSCC currents in response to the 1 s step depolarization are shown in A and B. Group data indicate that the normalized integrated L-type VSCC current (integral in pC divided by peak current; pC pA_peak⁻¹) is only moderately larger than the other two channel types in response to the 1 s depolarization (significant difference between the L- and N-type channels; P < 0.01 one-way ANOVA). For this analysis the currents were integrated over the 1 s waveform and normalized to the peak current (n = 17, 21 and 14 for L-, P/Q- and N-type respectively). C, voltage dependence of L-, P/Q- and N-type channel activation at 37 °C determined by monitoring the peak current response at the indicated potentials and expressing the data as a conductance change (g g_max⁻¹). The conductances were fit to the Boltzmann equation and the V_1/2 was determined. D, channel inactivation determined by a 10 ms test pulse from −100 to 0 mV given 2–5 ms after a 1 s inactivating step to 0 mV from the indicated holding potentials. The peak currents elicited at 0 mV were then normalized to the maximal response obtained and fitted to the Boltzmann equation to determine the voltage at which 50 % of the channels were inactivated: P/Q- (−64 mV, n = 5) and N-type Ca²⁺ channels (−64 mV, n = 8) have a markedly more negative inactivation profile than L-type channels (−35 mV, n = 7). E, step pulses of limiting duration indicate that APs effectively activate L-, P/Q- and N-type channels. A series of short step depolarizing pulses from −70 to +25 mV of varied duration from 0.1 to 3.1 ms were given to mimic APs of varying duration. An example of an overplotted L-type VSCC response to the short step pulses at 22 and 37 °C. A steady Ca²⁺ current as well as a large transient tail current was observed. F, group data showing the responses of all 3 channel types; open symbols indicate 22 °C responses and solid symbols 37 °C. The currents were normalized to the largest response of the series. α_1A (P/Q-type), α_1B (N-type) and α_1C (L-type) subunits were expressed in combination with β_1B and α_2δ subunits. Data shown are the means ± s.e.m. of data obtained from HEK 293 cells expressing L- (n = 8/22, 22/37 °C), P/Q- (n = 8/9) or N-type channels (n = 8/12).

Figure 3. Cumulative plots of Ca²⁺ currents in response to complex waveforms

A, voltage commands used to evoke the VSCC currents in HEK 293 cells. The inset shows the expanded view of the first firing segment. Calibration bar is 10 ms. B, sample cumulative plots of Ca²⁺ currents (total charge) recorded from individual HEK 293 cells expressing L-, P/Q- or N-type VSCCs (using the same current traces as shown in Fig. 2A). Responses were normalized to a preceding step pulse as described in the Methods (and employed in Fig. 3B) to compare the total charge influx mediated by the different VSCC types. C, cumulative current plots normalized to the maximal value permitting a better comparison of response kinetics. Note for N- and P/Q-type channels charge influx was primarily associated with APs. α_1A (P/Q-type), α_1B (N-type) and α_1C (L-type) subunits were expressed in combination with β_1B and α_2δ subunits.

Figure 4. β-Subunit expression differentially regulates the response of VSCCs to complex voltage waveforms

Inactivation was assessed by placing a 5 ms square pulse (to +25 mV) at the end of the train stimulus and comparing its response amplitude (tail Ca²⁺ current (I_Ca) peak) to the amplitude of the first action potential-induced current at 37 °C in both the repetitive firing and plateau waveforms. A, sample traces showing the VSCC current in response to the depolarization waveforms with the 5 ms end-pulses. α_1A (P/Q-type) and α_1C (L-type) subunits were expressed in combination with β_1B or β_2A (as indicated) and α_2δ subunits. B, group data showing the ratio of response amplitude (5 ms end-pulse response versus that to the first action potential). C, group data illustrating the normalized integrated current response for P/Q- and L-type channels expressing either β_1B or β_2A. *P < 0.05; **P < 0.01; ***P < 0.001, comparison for a particular channel and waveform with either β_1B or β_2A: L- (n = 9) and P/Q-type (n = 7).

Figure 5. Differential recovery of VSCCs from inactivation in response to a gap in AP firing

A, the left column shows the Ca²⁺ current in response to the repetitive firing waveform (see Fig. 4A for original), with four action potentials removed. During the AP gap (at −45 to −50 mV) the L-type VSCC current recovered while the P/Q- and N-type continued to inactivate. On the right column, the membrane potential during the gap was hyperpolarized to −140 mV leading to marked acceleration of P/Q- and N-type channel recovery from inactivation. B, group data illustrating differences in recovery between the channel types in response to the 4-AP gap. Values shown are ratio of the average amplitude of the Ca²⁺ current elicited by three action potentials after the gap versus average Ca²⁺ current amplitude for three action potentials before the gap. For L-type VSCCs, both the normal gap waveform (left column) and the −140 mV hyperpolarized gap waveform have significantly increased ratios (1.07 ± 0.03, n = 9 and 1.46 ± 0.16, n = 4 respectively) compared to normal repetitive firing waveform (0.99 ± 0.01, n = 9). For P/Q-type Ca²⁺ channels, removal of 4 APs did not significantly reduce the progressive inactivation (ratios: 0.82 ± 0.04, n = 5 and 0.87 ± 0.01, n = 5 for the regular repetitive waveform and normal gap waveform respectively). Hyperpolarization to −140 mV greatly accelerated the recovery of the P/Q-type channel from inactivation (3.26 ± 0.35, n = 8). This enhanced recovery is significantly more than that observed in the L-type channels (P = 0.0062). Similar to the P/Q-type channels, inactivation of the N-type was not attenuated in normal gap waveform (0.73 ± 0.02, n = 6) as compared to that in regular repetitive waveforms (0.75 ± 0.02, n = 8). The ratio for hyperpolarized gap waveform in N-type channels was also significantly increased (2.01 ± 0.28, n = 2). Experiment temperature was 37 °C. Asterisks indicate comparisons to the normal repetitive firing waveform among the same channel type: *P < 0.05; **P < 0.01; ***P < 0.001. α_1A (P/Q-type), α_1B (N-type) and α_1C (L-type) subunits were expressed in combination with β_1B and α_2δ subunits.

Figure 6. Differential effect of EPSP removal from complex waveforms on L-type versus P/Q- and N-type VSCCs

A, example of Ca²⁺ currents mediated by P/Q-type VSCCs stimulated by the repetitive firing voltage waveform without the EPSP component (centre panel) at 37 °C. Progressive inactivation of the VSCC current in response to AP stimuli was reduced by removal of EPSPs (see Fig. 2A for control example with both EPSPs and APs). Hyperpolarization to −110 mV (AP amplitude increased so the peaks are at the same membrane potential) produced a further attenuation of inactivation associated with the AP stimuli. B, group data showing the mean normalized integrated VSCC current in response to repetitive firing waveforms with different interspike membrane potentials (−70 or −110 mV for N-, L- and P/Q-type channels). α_1A (P/Q-type), α_1B (N-type) and α_1C (L-type) subunits were expressed in HEK 293 cells in combination with β_1B and α_2δ subunits. *P < 0.05; **P < 0.01; ***P < 0.001, comparison by ANOVA between with EPSP (typical repetitive firing waveform) and −70 mV no EPSP or −110 mV no EPSP, the results shown are from n = 4–12 cells for each group).

Figure 7. Recovery from inactivation induced by complex voltage waveforms

A, the repetitive firing voltage waveform as described in the previous figures, was preceded by a +25 mV control pulse and followed at varied intervals by a +25 mV test pulse. The current record on the right indicates how the control or test pulse evoked Ca²⁺ current was monitored at 37 °C. The point at which the current amplitude was measured (steady-state component, not tail current) is indicated by the arrow. B, plots of the normalized (to control pulse) recovery of test pulse amplitude (means ± s.e.m.) at varying intervals after the repetitive firing voltage waveform. The data was fitted to a single exponential function with an offset reflecting the degree of starting inactivation. The P/Q time constant (τ= 450 ± 77 ms, n = 6) was considerably longer than that derived for the L- (117 ± 24 ms, n = 5) or N-type (141 ± 21 ms, n = 5) VSCC. A minor slower component of recovery from inactivation with a τ > 2000 ms was also observed (≈20 %). α_1A (P/Q-type), α_1B (N-type) and α_1C (L-type) subunits were expressed in HEK 293 cells in combination with β_1B and α_2δ subunits.