Comparison of N- and P/Q-type voltage-gated calcium channel current inhibition

Abstract

Activation of N- and P/Q-type voltage-gated calcium channels triggers neurotransmitter release at central and peripheral synapses. These channels are targets for regulatory mechanisms, including inhibition by G-protein-linked receptors. Inhibition of P/Q-type channels has been less well studied than the extensively characterized inhibition of N-type channels, but it is thought that they are inhibited by similar mechanisms although possibly to a lesser extent than N-type channels. The aim of this study was to compare the inhibition of the two channel types. Calcium currents were recorded from adrenal chromaffin cells and isolated by the selective blockers omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (400 nM). The inhibition was elicited by ATP (100 microM) or intracellular application of GTP-gamma-S. It was classified as voltage-sensitive (relieved by a conditioning prepulse) or voltage-insensitive (present after a conditioning prepulse). The voltage-insensitive inhibition accounted for a 20% reduction of both currents, whereas the voltage-sensitive inhibition reduced the N-type current by 45% but the P/Q-type current by 18%. However, the voltage dependence of the inhibition, the time course of relief from inhibition during a conditioning prepulse, and the time course of reinhibition after such a prepulse showed few differences between the N- and P/Q-type channels. Assuming a simple bimolecular reaction, our data suggest that changes in the kinetics of the G-protein/channel interaction alone cannot explain the differences in the inhibition of the N- and P/Q-type calcium channels. The subtle differences in inhibition may facilitate the selective regulation of neurotransmitter release.

Fig. 1.

ATP inhibits N-type I_Cato a greater extent than P/Q-type I_Ca.A, Plotted is peak current amplitude against time. Cells were depolarized to +20 mV from a holding potential of −80 mV.ATP (100 μm) and toxins were applied to the cell, as indicated by the horizontal bars. The combination of ω-Cgtx GVIA (1 μm) andω-Aga IVA (400 nm) blocked almost all of the current in this cell. ATP inhibited both the ω-Cgtx GVIA and ω-Aga IVA-sensitive current components.B, N-type and P/Q-type I_Ca in the presence and absence (control) of ATP. TheN-type I_Ca currents are generated by subtracting the currents obtained after ω-Cgtx GVIA from those obtained before ω-Cgtx GVIA. The P/Q-type currents are that component of I_Ca that remained after block with ω-Cgtx GVIA. Note the slowed activation kinetics and the larger inhibition of the N-type current.C, The bar chart on the left plots the percentage of inhibition produced by 100 μm ATP in 35 cells like the one shown above (A, B). The percentage of inhibition of peak inward current amplitude is plotted for both the N- and P/Q-type currents. The currents were isolated by obtaining data before and after application of ω-Cgtx GVIA. There was a significantly larger inhibition of N-type, as compared with P/Q-type, current. The bar chart on the right plots data collected from nine cells in which ATP was applied before and afterω-Aga IVA. In this case the current after block withω-Aga IVA was termed the N-typeI_Ca, and the difference currents were P/Q-type I_Ca.

Fig. 2.

The magnitude of the voltage-sensitive block is different for the N- and P/Q-type currents. A, Shown are current records of N-type and P/Q-typeI_Ca isolated as in Figure 1. Thetraces labeled control were obtained in the absence of ATP, the traces labeledATP were obtained in the presence of 100 μm ATP, and the traces labeled ATP + prepulse were obtained in the continued presence of ATP but preceded by a depolarizing prepulse to + 100 mV lasting 50 msec. The voltage protocol is illustrated above the current records. The prepulse relieved ∼70% of the N-type current inhibition but only ∼50% of the P/Q-type current inhibition. B, The bar chart on the left plots the percentage of inhibition produced by 100 μm ATP for 17 cells like that in A. The inhibition is divided into the voltage-sensitive component (relieved by depolarizing prepulses) and the voltage-insensitive component (the inhibition that persists after a prepulse). The voltage-insensitive component was similar for the two currents, but the voltage-sensitive component was more than twice as large in the N-type current as in the P/Q-type current. The bar chart on the right illustrates that the same pattern of inhibition was seen if ω-Aga IVA (rather thanω-Cgtx GVIA) was used to isolate the currents. This single prepulse did not produce significant reversal of ω-Aga IVA block.

Fig. 3.

Intracellular GTP-γ-S mimics the inhibition elicited by ATP. A, Currents recorded from a cell with patch pipette solution containing GTP-γ-S. For these experiments one-fifth of the GTP in the patch pipette solution (total GTP = 350 μm) was replaced with an equal amount (70 μm) of GTP-γ-S. The currents on the leftwere recorded within 1 min of entering the whole-cell configuration before significant activation of G-proteins by the GTP-γ-S. Those on the right were recorded 7–8 min after entering the whole-cell configuration. Each trace shows two superimposed currents. The control current was elicited by a 20 msec test pulse to +20 mV with no prepulse (labeled no prepulse). The second current was preceded by a depolarizing prepulse to +100 mV for 50 msec, 10 msec before the test pulse (labeledwith prepulse). GTP-γ-S inhibitsI_Ca and slows the activation kinetics ofI_Ca in a manner indistinguishable from ATP. The dashed line is for illustrative purposes and represents the peak amplitude of the uninhibited current.B, Plotted is the percentage block ofI_Ca produced by 1 μmω-Cgtx GVIA for cells loaded with control or GTP-γ-S containing patch pipette solution. There was a significant reduction in toxin block when GTP-γ-S was present in the patch pipette solution.C, Shown is the potentiation of N- and P/Q-typeI_Ca amplitude produced by prepulses onGTP-γ-S (as shown in A) orATP (100 μm) inhibited currents. The increase in current amplitude produced by the prepulses was attributable to relief of the voltage-sensitive component of the inhibition. It is presented as the ratio of the current amplitude with a prepulse relative to that without a prepulse. The N-type current was potentiated significantly more than the P/Q-type current, reflecting the differential voltage sensitivity of the inhibition for the two channel types.

Fig. 4.

Time course of relief from inhibition is similar for N- and P/Q-type currents. The voltage protocol used is shown at thetop. The holding potential was −80 mV, and the test pulse was of 20 msec duration to +20 mV. The test pulse was preceded by a prepulse (to either +100 or +140 mV), and the two were separated by an interpulse of 10 msec during which the cell was returned to the holding potential. The patch pipette solution contained GTP-γ-S (70 μm) to elicit inhibition. The voltage protocols were repeated before and after application of ω-Cgtx GVIA (1 μm) to isolate the N- and P/Q type currents.A, Plotted is relief from inhibition as a function of prepulse duration by prepulses to +100 mV. The increase in current amplitude produced by each of the prepulses (i.e., the amplitude of the current with a prepulse minus the amplitude of a current without a prepulse) was normalized to that produced by the longest duration prepulse (50 msec), which is known to produce a maximal relief from inhibition. The data were fit with a single exponential, giving time constants of 9.8 ± 0.7 msec for the N-type current and 13.1 ± 1.0 msec for the P/Q-type current.B, Plotted is relief from inhibition by prepulses to +140 mV. Time constants were 11.5 ± 1.0 msec forN-type current and 11.7 ± 0.8 msec forP/Q-type current.

Fig. 5.

Time course of reinhibition is similar for N- and P/Q-type currents. The voltage protocol used is shown at thetop. The holding potential was −80 mV, and the test pulse was of 20 msec duration to +20 mV. The test pulse was preceded by a prepulse of 50 msec duration to +100 mV, and the two were separated by an interpulse during which the cell was repolarized to either −60 mV or −80 mV. The duration of the interpulse was varied from 5 to 300 msec. The patch pipette solution contained GTP-γ-S (70 μm) to elicit inhibition. The voltage protocols were repeated before and after application of ω-Cgtx GVIA (1 μm) to isolate the N- and P/Q type currents.A, Plotted is reinhibition as a function of interpulse duration at −60 mV. The increase in current amplitude caused by each of the prepulses was normalized to that produced by a prepulse followed by the shortest duration of interpulse (5 msec). The data were fit with a single exponential, giving time constants of reinhibition of 108 ± 11 msec for the N-type and 105 ± 11 msec for the P/Q-type currents. B, Plotted is reinhibition at −80 mV. The time constants of the reinhibition were 112 ± 17 msec for the N-type current and 91 ± 8 msec for the P/Q-type current.

Fig. 6.

Comparison of the voltage dependence of the relief from inhibition for N- and P/Q-type channels. The voltage protocol used is shown at the top. The holding potential was −80 mV, and the test pulse was to +20 mV for 20 msec. The duration of the prepulse and interpulse was not varied, and both were 10 msec. The potential of the prepulse was varied from −10 to +120 mV. The patch pipette contained GTP-γ-S (70 μm) to elicit the inhibition. Voltage protocols were repeated before and after application of ω-Cgtx GVIA (1 μm) to isolate theN-type and P/Q type currents. The graph plots data from six cells in which the increase in amplitude produced by each of the prepulses was normalized to the increase produced by the most positive prepulse (+120 mV). The data were plotted against prepulse potential and fit with a sigmoidal curve.

Fig. 7.

Voltage dependence of N- and P/Q-type calcium current I/V curves. A, ThreeI/V curves are superimposed. The first (control) was obtained before toxin application and so is composed of both N- and P/Q-typeI_Ca. The second was obtained after block by ω-Cgtx GVIA (1 μm) and thus represents mainly the P/Q-type current (along with the small toxin-insensitive component). Note that in addition to a reduction in amplitude the ω-Cgtx GVIA shifted the I/V curve to the left. The third curve was obtained by subtracting the curve obtained after ω-Cgtx GVIA from that obtained before ω-Cgtx GVIA. This differenceI/V represents the pure N-type current and is shifted to the right relative to control. Note that the shift in reversal potential was apparent only in some cells and was attributable, at least in part, to a small residual current present in some cells after leak subtraction. B, In this set of experiments ω-Agatoxin IVA (400 nm) was used to block and isolate P/Q-type I_Ca. After block with ω-Aga IVA, the I/Vcurve shifted to the right, and the differenceI/V shifted to the left relative to control. Both manipulations reveal that the P/Q-type I/Vcurve shifted by ∼7 mV to the left relative to the N-type I/V curve.