Independent and joint modulation of rat Nav1.6 voltage-gated sodium channels by coexpression with the auxiliary β1 and β2 subunits

Abstract

The Na(v)1.6 voltage-gated sodium channel α subunit isoform is the most abundant isoform in the brain and is implicated in the transmission of high frequency action potentials. Purification and immunocytochemical studies imply that Na(v)1.6 exist predominantly as Na(v)1.6+β1+β2 heterotrimeric complexes. We assessed the independent and joint effects of the rat β1 and β2 subunits on the gating and kinetic properties of rat Na(v)1.6 channels by recording whole-cell currents in the two-electrode voltage clamp configuration following transient expression in Xenopus oocytes. The β1 subunit accelerated fast inactivation of sodium currents but had no effect on the voltage dependence of their activation and steady-state inactivation and also prevented the decline of currents following trains of high-frequency depolarizing prepulses. The β2 subunit selectively retarded the fast phase of fast inactivation and shifted the voltage dependence of activation towards depolarization without affecting other gating properties and had no effect on the decline of currents following repeated depolarization. The β1 and β2 subunits expressed together accelerated both kinetic phases of fast inactivation, shifted the voltage dependence of activation towards hyperpolarization, and gave currents with a persistent component typical of those recorded from neurons expressing Na(v)1.6 sodium channels. These results identify unique effects of the β1 and β2 subunits and demonstrate that joint modulation by both auxiliary subunits gives channel properties that are not predicted by the effects of individual subunits.

Figure 1

Sodium currents carried by Na_v1.6, Na_v1.6+β1, Na_v1.6+β2, and Na_v1.6+β1+β2 sodium channels. (A) Four representative sodium current traces recorded from oocytes expressing Na_v1.6, Na_v1.6+β1, Na_v1.6+β2, or Na_v1.6+β1+β2 sodium channels and a single trace from the oocyte expressing Na_v1.6 sodium channels after the bath application of 300 nM TTX. Peak transient currents in the absence of TTX (~2 – 2.5 μA) were scaled to facilitate visual comparison of decay kinetics. (B) Comparison of the fast and slow kinetic constants for peak current decay obtained from experiments such as those shown in Panel A. Values (means ± SE of 16–44 separate experiments with different oocytes) marked with different letters were significantly different by one-way ANOVA with Tukey’s multiple comparison test (P < 0.05). (C) Voltage dependence of persistent currents measured at the end of 40-ms test depolarizations from −100 mV to the indicated potentials in oocytes expressing Na_v1.6, Na_v1.6+β1, Na_v1.6+β2 or Na_v1.6+β1+β2 sodium channels. Values are means of 16–44 separate experiments with different oocytes; bars show SE values larger than the data point symbols. Positive values represent inward currents. The arrow indicates the test potential employed in the experiments shown in Panel A.

Figure 2

Voltage-dependent activation and steady-state inactivation of Na_v1.6, Na_v1.6+β1, Na_v1.6+β2, and Na_v1.6+β1+β2 sodium channels. (A) Conductance-voltage plots for channel activation. Peak sodium currents were obtained obtained using the indicated pulse protocol were transformed to conductances (G) using the equation G = I/(V_t-V_rev), where I is the peak current, V_rev is the reversal potential, and V_t is the voltage of the test potential; conductances were then normalized to the maximum conductance (G_max) for that oocyte. Values are means of 15–48 separate experiments with different oocytes; bars show SE values larger than the data point symbols. Curves were fitted to the mean values using the Boltzmann equation. (B) Voltage dependence of steady-state inactivation. Amplitudes of peak transient currents obtained using the indicated pulse protocol are plotted as a function of prepulse potential (V_p). Values are means of 11–34 separate experiments with different oocytes; bars show SE values larger than the data point symbols. Curves were fitted to the mean values using the Boltzmann equation.

Figure 3

Effect of repeated depolarization on the stability of sodium currents recorded from oocytes expressing Na_v1.6, Na_v1.6+β1, Na_v1.6+β2, or Na_v1.6+β1+β2 sodium channels. Sodium currents were recorded during a 40-ms step depolarization from −100 mV to 0 mV following 0–100 conditioning prepulses (5-ms pulses from −100 mV to 10 mV at 66.7 Hz). Values are means of 11–31 separate experiments with different oocytes; bars show SE values larger than the data point symbols.