Kv2 channel regulation of action potential repolarization and firing patterns in superior cervical ganglion neurons and hippocampal CA1 pyramidal neurons

Abstract

Kv2 family "delayed-rectifier" potassium channels are widely expressed in mammalian neurons. Kv2 channels activate relatively slowly and their contribution to action potential repolarization under physiological conditions has been unclear. We explored the function of Kv2 channels using a Kv2-selective blocker, Guangxitoxin-1E (GxTX-1E). Using acutely isolated neurons, mixed voltage-clamp and current-clamp experiments were done at 37°C to study the physiological kinetics of channel gating and action potentials. In both rat superior cervical ganglion (SCG) neurons and mouse hippocampal CA1 pyramidal neurons, 100 nm GxTX-1E produced near-saturating block of a component of current typically constituting ∼60-80% of the total delayed-rectifier current. GxTX-1E also reduced A-type potassium current (IA), but much more weakly. In SCG neurons, 100 nm GxTX-1E broadened spikes and voltage clamp experiments using action potential waveforms showed that Kv2 channels carry ∼55% of the total outward current during action potential repolarization despite activating relatively late in the spike. In CA1 neurons, 100 nm GxTX-1E broadened spikes evoked from -70 mV, but not -80 mV, likely reflecting a greater role of Kv2 when other potassium channels were partially inactivated at -70 mV. In both CA1 and SCG neurons, inhibition of Kv2 channels produced dramatic depolarization of interspike voltages during repetitive firing. In CA1 neurons and some SCG neurons, this was associated with increased initial firing frequency. In all neurons, inhibition of Kv2 channels depressed maintained firing because neurons entered depolarization block more readily. Therefore, Kv2 channels can either decrease or increase neuronal excitability depending on the time scale of excitation.

Keywords: Guangxitoxin; Hodgkin-Huxley kinetics; Kv2; activation; deactivation; delayed-rectifier potassium channel.

Figure 1.

Effect of GxTX-1E on voltage-activated currents in rat SCG neurons. A, Effect of 100 and 200 nm GxTX-1E on outward currents in a rat SCG neuron evoked by steps from −90 mV to +30 mV (left) and −70 mV to +30 mV (right). The membrane was held at −70 mV for 1 s between the two steps to +30 mV. B, Effect of 100 and 200 nm GxTX-1E on peak outward current evoked from −90 mV (same cell as in A). C, Effect of 100 and 200 nm GxTX-1E on steady-state outward current (measured at the end of 200 ms steps) evoked from −90 mV (same cell as in A, B). D, Effect of GxTX-1E on I_A evoked by a step from −90 to −40 mV. E, Effect of GxTX-1E on sodium current evoked by a step from −90 mV to −10 mV. Recording made with internal Cs-based solution and external solution containing 25 mm NaCl, 2 mm CaCl₂, and 128.5 TEA Cl. Initial fast sodium current is followed by a small sustained calcium current. F, Calcium current evoked by a step from −50 mV to −10 mV before and after application of 100 nm GxTX-1E. Same solutions as in E; cell was held at −50 mV to inactivate sodium current.

Figure 2.

Voltage dependence of Kv2 current determined by GxTX subtraction in rat SCG neurons. A, GxTX-sensitive current determined by subtracting currents recorded before and after applying 200 nm GxTX-1E. Solution contained 1 μm TTX, 1 μm ω-conotoxin GVIA, and 1 mm TEA. B, Voltage dependence of activation of GxTX-sensitive current determined by plotting tail current at −50 mV as a function of the test voltage. Solid red line, Fit to simple Boltzmann function 1/(1 + exp − (V − V_h)/k), where V_h = −12.9 mV is the midpoint and k = 6.2 mV is the slope factor. Solid blue line: fit to a Boltzmann function raised to fourth power, [1/(1 + exp − (V − V_hn)/k_n)]⁴, where V_hn = −27.4 mV is the voltage at which half of the n particles are in the activated position and k_n = 8.6 mV is the slope factor for activation of n particles. C, Mean ± SEM for normalized conductance determined by tail current activation curves in SCG neurons (n = 7). Solid red line: simple Boltzmann function 1/(1 + exp − (V − V_h)/k), where V_h = −13.1 mV and k = 6.3 mV are mean values of results of separate fits in each of 7 cells (SEM values of 0.9 and 0.1 mV, respectively).

Figure 3.

Activation and deactivation kinetics of Kv2 current in rat SCG neurons. A, Activation kinetics of Kv2 current isolated by GxTX subtraction as in Figure 2. Activation was fit by n⁴ kinetics, A[1 − exp(−t/τ_n)]⁴, where A is steady-state amplitude and τ_n is the time constant for n particle gating. Fits are shown as red lines. B, Kinetics of deactivation. Red lines show fits to n⁴ kinetics, A[n_{∞ −} (n_∞ − n₀)exp(−t/τ_n))]⁴, where A is an amplitude factor, n₀ and n_∞ are the starting and final values of n, respectively, and τ_n is the time constant for n particle gating. C, τ_n as a function of voltage. Mean ± SEM for determinations in seven cells for activation (open circles) and seven cells for deactivation (filled squares). Blue line, τ_n for delayed-rectifier current in squid axon at 6.3°C from Hodgkin-Huxley equations (Hodgkin and Huxley, 1952). Green line, τ_n for delayed-rectifier current in squid axon at 37°C calculated using a Q₁₀ of 3. D, Dominant time constants for activation (open circles) and deactivation (filled squares) in SCG neurons obtained by fitting the major relaxation of current by a single exponential. Activation was fit starting at 35% of the rise (to avoid the lag) and deactivation was fit to the final 80% of the decay.

Figure 4.

Effect of GxTX-1E on repetitive action potential firing in a rat SCG neuron. A, Effect of 100 nm GxTX-1E on firing evoked by a 500 ms 50 pA current injection. B, Effect of 100 nm GxTX-1E on the width of the first action potential (measured at midpoint) during firing evoked by 50 pA current injections. Connected gray circles show changes in individual cells; connected black circles show mean values ± SEM. Average spike width was 2.12 ± 0.08 ms in control and 2.94 ± 0.17 ms in 100 nm GxTX-1E (n = 26; p = 1.7 × 10⁻⁷). C, Effect of 100 nm GxTX-1E on initial firing frequency (measured from first two spikes) for 500 ms current injections. D, Effect of 100 nm GxTX-1E on number of spikes (defined as having amplitude >40 mV) in the 500 ms current injection period. E, Effect of 100 nm GxTX-1E on membrane potential of the first trough.

Figure 5.

Effect of GxTX-1E on ionic currents during action potentials in a rat SCG neuron. A, Effect of 100 nm GxTX-1E on ionic currents evoked by action potential waveforms in voltage-clamp mode. Firing in response to a current injection of 50 pA was recorded and then used as the command waveform in voltage clamp. Current evoked by the waveform was signal averaged over four sweeps before (black) and after (red) application of 100 nm GxTX-1E. Capacitative current was nulled using the capacity compensation circuitry of the amplifier. B, Action potential-evoked currents for the first (left) and third (right) action potentials shown at faster time base. Inward sodium currents are truncated. Black, Before application of GxTX-1E. Red, After GxTX-1E. Blue, Point by point subtraction of before-after to obtain GxTX-sensitive current.

Figure 6.

Effect of GxTX-1E on voltage-activated outward currents in mouse hippocampal CA1 neurons. A, Effect of 30, 100, and 200 nm GxTX-1E on currents evoked by a step from −90 mV to +10 mV. B, Time course of inhibition. Current was measured at the end of a 200 ms step to +10 mV as in A. C, Collected results for inhibition of current evoked by a step from −90 mV to +10 mV (mean ± SEM, n = 11 for 30 nm GxTX-1E, n = 10 for 100 and 200 nm GxTX-1E; p < 10⁻⁶ for all concentrations relative to control; p = 0.02 for difference between 100 and 200 nm).

Figure 7.

Voltage dependence of Kv2 current determined by GxTX subtraction in mouse CA1 pyramidal neurons. A, GxTX-sensitive (red) and GxTX-resistant (blue) current determined by application of 100 nm GxTX-1E. Solution contained 1 μm TTX. B, Voltage dependence of activation of peak GxTX-sensitive conductance from the cell in A. Peak conductance was calculated from peak current using a reversal potential of −99 mV, the calculated potassium equilibrium potential with the solutions used. Solid red line, Fit to simple Boltzmann function 1/(1 + exp − (V − V_h)/k), where V_h = −10.1 mV is the midpoint and k = 10.7 mV is the slope factor. Solid blue line, Fit to a Boltzmann function raised to fourth power, [1/(1 + exp − (V − V_hn)/k_n)]⁴, where V_hn = −34.9 mV is the voltage at which half of the n particles are in the activated position and k_n = 15.1 mV is the slope factor for activation of n particles. C, Mean ± SEM for normalized conductance in 20 CA1 neurons. Solid red line, Boltzmann function 1/(1 + exp − (V − V_h)/k), where V_h = −11.2 mV and k = 10.4 mV are mean values of results of separate fits in each cell (SEM values of 1.3 and 0.5 mV, respectively). Average maximal conductance was 48 ± 7 nS (n = 20).

Figure 8.

Activation and deactivation kinetics of Kv2 current in mouse CA1 pyramidal neurons. A, Activation kinetics of Kv2 current isolated by GxTX subtraction with 100 nm GxTX-1E. Red lines, Single exponential fit to rising phase of current fit from 35% to maximal current. B, Kinetics of deactivation at −50 mV. Red line, Fit to a single exponential. C, Time constants from single exponential fits as a function of voltage. Mean ± SEM for determinations in 20 CA1 neurons (blue symbols) compared with measurements made in the same way in seven SCG neurons (red symbols). Activation was fit starting at 35% of the rise (to avoid the lag) and deactivation was fit to the final 80% of the decay.

Figure 9.

Effect of GxTX-1E on action potentials in mouse hippocampal CA1 neurons. A, Effect of 100 nm GxTX-1E on action potentials evoked by a 40 pA current injection from a membrane potential near −80 mV. B, Effect of 100 nm GxTX-1E on action potentials evoked by a 90 pA current injection from a membrane potential near −70 mV. The action potential with 100 nm GxTX-1E activated slightly earlier due to a slightly (∼1 mV) more depolarized membrane potential; to better compare the shapes, it has been shifted by 160 μs so that the rising phases align. C, Effect of 100 nm GxTX-1E on the width of action potentials evoked from near −80 mV or −70 mV. Width was measured at half-amplitude of the action potential measured from the depolarizing phase. Measurements were made for action potentials evoked by a current injection of 50 pA. Connected gray circles show changes in individual cells; connected black circles show mean values. For −80 mV data, control mean was 0.83 ± 0.04 ms and GxTX mean was 0.89 ± 0.04 ms, n = 7; p = 0.12. Average V_m before current injection was −79.5 ± 0.8 mV. For −70 mV data, control mean was 1.02 ± 0.06 ms and GxTX mean was 1.30 ± 0.08 ms; n = 7, p = 0.005. Average V_m before current injection was −68.2 ± 0.5 mV. D, Effect of 100 nm GxTX-1E on the trough after the action potential for action potentials evoked from near −80 mV or −70 mV (same action potentials as in C). Trough was measured at the most negative voltage after the spike. Measurements were made for action potentials evoked by a current injection of 50 pA. Connected gray circles show changes in individual cells; connected black circles show mean values. For −80 mV data, control mean was −74.9 ± 1.7 mV and GxTX mean was −66.3 ± 2.1 mV, n = 7; p = 0.005. For −70 mV data, control mean was −75.8 ± 1.2 mV and GxTX mean was −64.7 ± 2.4 mV; n = 7, p = 0.001.

Figure 10.

Effect of GxTX-1E on repetitive firing in a mouse hippocampal CA1 neuron. A, Effect of 100 nm GxTX-1E on repetitive firing elicited by a 70 pA current injection for 500 ms. B, Initial firing frequency (calculated from first two spikes) as a function of injected current. C, Number of spikes in the 500 ms current injection period (with a height criterion of 40 mV). D, Most negative voltage (trough) between first and second spikes. E, Average membrane potential during the current injection.