Inwardly permeating Na ions generate the voltage dependence of resurgent Na current in cerebellar Purkinje neurons

Abstract

Voltage-gated Na channels of cerebellar Purkinje neurons express an endogenous open-channel blocking protein. This blocker binds channels at positive potentials and unbinds at negative potentials, generating a resurgent Na current and permitting rapid firing. The macroscopic voltage dependence of resurgent current raises the question of whether the blocker directly senses membrane potential or whether voltage dependence is conferred indirectly. Because we previously found that inwardly permeating Na ions facilitate dissociation of the blocker, we measured voltage-clamped currents in different Na gradients to test the role of permeating ions in generating the voltage dependence of unblock. In reverse gradients, outward resurgent currents were tiny or absent, suggesting that unblock normally requires "knockoff" by Na. Inward resurgent currents at strongly negative potentials, however, were larger in reverse than in control gradients. Moreover, occupancy of the blocked state was prolonged both in reverse gradients and in control gradients with reduced Na concentrations, indicating that block is more stable when inward currents are small. Accordingly, reverse gradients shifted the voltage dependence of block, such that resurgent currents were evoked even after conditioning at negative potentials. Additionally, in control gradients, peak resurgent currents decreased linearly with driving force during the conditioning step, suggesting that the stability of block varies directly with inward Na current amplitude. Thus, the voltage dependence of blocker unbinding results almost entirely from repulsion by Na ions occupying the external pore. The lack of voltage sensitivity of the blocking protein suggests that the blocker's binding site lies outside the membrane field, in the permeation pathway.

Figure 1.

Reverse Na gradients reduce outward resurgent currents. A, Voltage protocol and representative traces of resurgent currents. Inset, Outward resurgent current at −30 mV in reverse gradient at high gain. B, Mean resurgent conductance versus voltage for control (n = 17), near-symmetric (n = 10), and reverse (n = 8) gradients. C, Resurgent current rise time versus voltage for control (n = 16), reverse (n = 8), and near-symmetric (n = 10) gradients. Symbols as in B. D, Voltage protocol and normalized representative traces (top). All traces are plotted upward for comparison. Bottom, mean rise time (left) and decay τ (right) for control (n = 17), reverse (n = 8), and near-symmetric (n = 7) gradients.

Figure 2.

Reverse Na gradients differentially affect transient and resurgent Na currents. A, Voltage protocol and representative traces for Na current activation. B, Activation curves with mean fit parameters for control and reverse: V_1/2 = −34.3, −30.7 mV; k = 5.1, 8.8 mV; G_max = 122, 92 nS; n = 16, 8. Conductance is normalized (top) for comparison of V_1/2 and k, and raw (bottom) for comparison of G_max. C, Conductance ratio of reverse to control gradients for predictions by the GHK current equation, and measured transient and resurgent conductances. Shaded area indicates voltages below reversal potential in reverse gradients.

Figure 3.

The blocker binds channels stably in reverse Na gradients. A, Voltage protocol and representative resurgent currents after 5–50 ms conditioning steps (left). Normalized resurgent currents versus conditioning duration for control (n = 6), reverse (n = 9), and reduced control (n = 9, right). B, Voltage protocol and representative transient currents at 0 mV to assess slow inactivation after 5–50 ms conditioning steps (left). The protocol is an extension of that in A. Normalized transient currents versus conditioning duration for control (n = 6), reverse (n = 7), and reduced control (n = 7).

Figure 4.

The blocker requires knockoff by inwardly permeating Na to unbind. A, Voltage protocol (top) and representative traces (bottom and middle at high gain). B, Mean fractional resurgent currents versus voltage for control (n = 9) and reverse (n = 6) gradients. Activation curves of transient current (Fig. 2) are replotted for comparison.