Differential facilitation of N- and P/Q-type calcium channels during trains of action potential-like waveforms

Abstract

Inhibition of presynaptic voltage-gated calcium channels by direct G-protein betagamma subunit binding is a widespread mechanism that regulates neurotransmitter release. Voltage-dependent relief of this inhibition (facilitation), most likely to be due to dissociation of the G-protein from the channel, may occur during bursts of action potentials. In this paper we compare the facilitation of N- and P/Q-type Ca(2+) channels during short trains of action potential-like waveforms (APWs) using both native channels in adrenal chromaffin cells and heterologously expressed channels in tsA201 cells. While both N- and P/Q-type Ca(2+) channels exhibit facilitation that is dependent on the frequency of the APW train, there are important quantitative differences. Approximately 20 % of the voltage-dependent inhibition of N-type I(Ca) was reversed during a train while greater than 40 % of the inhibition of P/Q-type I(Ca) was relieved. Changing the duration or amplitude of the APW dramatically affected the facilitation of N-type channels but had little effect on the facilitation of P/Q-type channels. Since the ratio of N-type to P/Q-type Ca(2+) channels varies widely between synapses, differential facilitation may contribute to the fine tuning of synaptic transmission, thereby increasing the computational repertoire of neurons.

Figure 1. Trains of APWs partially relieve the inhibition of I_Ca in adrenal chromaffin cells

Chromaffin cells were stimulated with trains of 20 APWs applied at 45 Hz in the absence (control) or in the presence of 100 μm ATP. To determine the maximum voltage-dependent facilitation, a single APW was preceded by a conditioning prepulse to +100 mV for 50 ms. A, the upper left panel shows the voltage commands applied to the cell and the lower left panel shows the currents elicited by those voltage stimuli. An APW train was applied before (labelled ‘control’), and during application of 100 μm ATP (labelled ‘ATP’). ‘P’ indicates the I_Ca activated by a single APW preceded by a prepulse in the continued presence of ATP (the outward current elicited by the prepulse is also shown, but note that the tail current has been suppressed). The dashed lines indicate the amplitude of I_Ca for the 1st step within the APW trains and the amplitude of the current following a prepulse. The prepulse protocol was used to distinguish between voltage-dependent (marked ‘VD’) and voltage-independent (marked ‘VI’) inhibition as detailed in the text. The panel to the right illustrates an individual APW and leak subtracted I_Ca on an expanded time scale. B, mean facilitation produced by each APW during a train under control conditions and with ATP present. Facilitation was calculated by normalizing the peak amplitude of I_Ca to the first current within each train. C, the peak facilitation produced by a prepulse, the train of APWs and the train of APWs corrected for the decrease seen under control conditions. Note, the dashed line represents the theoretical facilitation that would be achieved if all the inhibition produced by ATP were reversed.

Figure 3. Differential facilitation of recombinant α_1A and α_1B channels by APW trains

Currents were recorded from tsA201 cells transiently transfected with either α_1A or α_1B along with β_2a and α₂δ subunits. GTPγS was included in the intracellular solution to tonically activate G-proteins and inhibit I_Ca. A, α_1A channel currents (left) or α_1B channel currents (right) in response to a train of 20 ‘normal’ APWs applied at 45 Hz. Following the train (indicated by ‘P’) is a current elicited by a single APW that was preceded by a conditioning prepulse of +100 mV for 50 ms. (Note that the outward current elicited during the prepulse is not shown.) The current amplitudes have been scaled to aid in comparison (note the different scale bars). Dashed lines indicate the amplitude of the first current within the train and the amplitude of the current preceded by a prepulse. B, the mean peak facilitation produced by the prepulse and train for both channel types. Note the difference in the ordinate.

Figure 4. Shortening the APW duration has greater effects on N-type I_Ca facilitation than on P/Q-type I_Ca facilitation

Cells were stimulated with a train of 20 ‘normal’ APWs at 45 Hz (see Fig. 1 for details) and then by a train of 20 ‘brief’ APWs at 45 Hz (the duration of the repolarization phase of the ‘brief’ APWs was digitally shortened as illustrated in the inset below panel A). A, the facilitation produced by trains in pharmacologically isolated P/Q-type (left) and N-type (right) Ca²⁺ channels from adrenal chromaffin cells. ATP (100 μm) was present to inhibit I_Ca. B, the facilitation of α_1A (left) and α_1B (right) type channels (coexpressed with β_2a and α₂δ in tsA201 cells). Inhibition of I_Ca was produced by including GTPγS in the intracellular solution. C, the mean peak facilitation produced by the ‘normal’ APW train (filled bars) and the ‘brief’ APW train (hatched bars) for each of the two channel types. P/Q-type/α_1A channels are shown on the left and N-type/α_1B channels are shown on the right. * Statistical significance between the ‘normal’ and ‘brief’ trains (P ≤ 0.001).

Figure 5. Dependence of facilitation on the frequency of APW trains

tsA201 cells were transiently transfected with either α_1A or α_1B channels (along with β_2a and α₂δ) and inhibition was elicited by inclusion of GTPγS in the intracellular pipette solution. Cells were stimulated by multiple trains of 20 APWs (either ‘normal’ or ‘brief’) at various frequencies (see inset below panel A for APW parameters). A, the facilitation produced by ‘normal’ APW trains applied at frequencies of 5–45 Hz. B, the facilitation produced by ‘brief’ APW trains applied at frequencies of 5–83 Hz. C, peak facilitation is plotted against train frequency for ‘normal’ and ‘brief’ APW trains. Linear regression fits to the data are shown.

Figure 2. Differential facilitation of native N- and P/Q-type I_Ca by APW trains

N- and P/Q-type I_Ca were pharmacologically isolated in adrenal chromaffin cells using selective channel blockers. A, the mean facilitation for each I_Ca within a train of 20 APWs applied at 45 Hz under control conditions (before application of ATP) or when I_Ca was inhibited (ATP present). The graph on the left shows isolated P/Q-type channels (the current component remaining after block with 1–2 μm ω-conotoxin GVIA). The graph on the right shows the data for isolated N-type channels (the current component remaining after block with 100–300 nm ω-Agatoxin IVA). Control currents showed a progressive decrease in amplitude whereas inhibited currents showed facilitation (1.19 ± 0.02, n = 12 for N-type and 1.15 ± 0.02, n = 12 for P/Q-type). B, the peak facilitation produced for each channel type by a prepulse, the train of APWs and the train of APWs corrected for the decrease seen under control conditions. The dashed lines represent the theoretical level of facilitation if all the inhibition produced by ATP was reversed. Note the difference in the ordinates.

Figure 6. The facilitation of α_1B channels shows a greater voltage dependence than do α_1A channels

tsA201 cells were transiently transfected with α_1A or α_1B channels (along with β_2a and α₂δ subunits) and inhibition was elicited by inclusion of GTPγS in the intracellular pipette solution. Cells were stimulated by multiple trains applied at 45 Hz in which the peak voltage of the APW was changed in 5 mV increments from +15 mV to +45 mV (see inset to panel A). A, the facilitation produced by trains of ‘normal’ APWs. Changing the peak voltage of the APW had no effect on the facilitation of α_1A channels (left) but greatly altered the facilitation of α_1B channels (right). B, the mean peak facilitation produced by each train plotted against the peak voltage of the APW for that train. The filled bars are for ‘normal’ APWs (* statistical significance relative to +45 mV; P < 0.004). Cells were also stimulated with ‘brief’ APW trains, shown by the hatched bars (# statistical significance relative to +45 mV; P < 0.01).