Resurgent current in context: Insights from the structure and function of Na and K channels

Abstract

Discovered just over 25 years ago in cerebellar Purkinje neurons, resurgent Na current was originally described operationally as a component of voltage-gated Na current that flows upon repolarization from relatively depolarized potentials and speeds recovery from inactivation, increasing excitability. Its presence in many excitable cells and absence from others has raised questions regarding its biophysical and molecular mechanisms. Early studies proposed that Na channels capable of generating resurgent current are subject to a rapid open-channel block by an endogenous blocking protein, which binds upon depolarization and unblocks upon repolarization. Since the time that this mechanism was suggested, many physiological and structural studies of both Na and K channels have revealed aspects of gating and conformational states that provide insights into resurgent current. These include descriptions of domain movements for activation and inactivation, solution of cryo-EM structures with pore-blocking compounds, and identification of native blocking domains, proteins, and modulatory subunits. Such results not only allow the open-channel block hypothesis to be refined but also link it more clearly to research that preceded it. This review considers possible mechanisms for resurgent Na current in the context of earlier and later studies of ion channels and suggests a framework for future research.

Figure 1

Resurgent Na current in Purkinje cells. (A) Activation and inactivation of TTX-sensitive transient Na currents of Purkinje cells dissociated from rat pups. Left: currents evoked by 50-ms step depolarizations from −90 mV to potentials ranging from −80 to +50 mV (10-mV steps). Right: conductance-voltage curve for activation and availability curve for steady-state inactivation after 100-ms conditioning steps ((2), with permission; copyright 1997, Society for Neuroscience). (B) The first published recordings of resurgent current of Purkinje cells. Left: TTX-sensitive Na currents evoked by repolarizations from −60 to −20 mV (10-mV steps) after a 10-ms depolarization to +30 mV. Right: current-voltage relation for peak resurgent current ((2), with permission; copyright 1997, Society for Neuroscience). (C) Experimentally recorded resurgent current from a Purkinje cell from an adult mouse ((126), with permission) with superimposed traces simulated by a Markov model with resurgent current generated by permeation-dependent open-channel block and unblock ((84), with permission). (D) Resurgent current evoked by repolarization to −30 mV ((8), with permission), with C, O, X+, and X− states labeled.

Figure 2

Relation of VSD_IV to activation, inactivation, and recovery. (A) VSD_IV deployment is unnecessary for activation. Fluorescence signals with outward movement of VSD_IV superimposed on outward ionic current through Na_V1.4 channels evoked by depolarization. Arrows emphasize that current activation precedes outward movement of VSD_IV ((25), with permission). (B) VSD_IV deployment is sufficient for inactivation. Steady-state inactivation assayed by transient Na currents evoked by depolarizations after conditioning at different voltages in wild-type Na_V1.4 and channels mutated (CN) to stabilize each VSD (DI, DII, DIII, DIV) in the outward position. Top: sample traces. Bottom: steady-state inactivation curves (mean ± SEM), indicating that fast inactivation is unaffected by outward VSDs I, II, or III, but shifted to negative (arrow) with outward movement of VSD_IV ((26), with permission). (C and D) Recovery current does not flow upon repolarization. The complete decay of Na current (arrows) upon repolarization to −80 mV after a 1-ms or 15-ms depolarization to 0 mV in a squid axon (C) ((35), with permission), and upon repolarization to the voltages indicated, in rat CA1 hippocampal neurons (D) ((36), with permission).

Figure 3

Open-channel block as a basis for hooked tail currents. (A) Resurgent current-like hooked tail currents (arrow) appear with inward flux of K⁺ ions through squid axon K channels inactivated by quaternary ammonium ion. Left: normal artificial sea water (ASW). Right: high external Na (440 mM) ((51), with permission). (B) Time course of hooked tail current carried by K⁺ flowing inward upon repolarization (diamonds) matches that of recovery from N-type inactivation (squares) in Shaker K channels ((53), with permission). (C) Quaternary ammonium ions and the inactivating N-terminus bind in the K channel pore. Representation of the KcsA channel (blue) crystallized with the inactivating quaternary ammonium ion tetrabutylammonium (red) ((57), with permission). (D) Intracellular exogenous blockers applied to squid axons accelerate inactivation of Na currents upon depolarization (top, arrows) and generate hooked tail currents upon repolarization (bottom, arrows). Left: Pancuronium ion ((63), with permission); middle: N-methyl-strychnine ((64), with permission); right: azure A ((35), with permission). (E) Cryo-EM structure of quinidine (blue) in the Na_V1.5 channel pore. Note the binding of the IFM sequence of the III-IV linker outside the pore (arrow) ((66,67), with permission). (F) Drug-binding sites in the pore domain of Na_V1.7. Site E, μ-conotoxin; site S, TTX; site C, quinidine, propafenone, and cannabidiol; site BIG (beneath the intracellular gate), carbamazepine, lacosamide, and bupivacaine. Site I indicates binding site of the fast-inactivation linker, outside the permeation pathway. F, fenestrations; G, intracellular gate ((66,67), with permission).

Figure 4

Interactions between fast inactivation and the open-channel blocked X+ state. (A) Intracellular proteolysis has the reverse effect of adding an exogenous open-channel blocker. Purkinje Na currents from an inside-out patch before (black) and after (gray) brief exposure to trypsin. With only classical fast inactivation remaining, transient currents are slowed (left) and resurgent current is removed (right) ((70), with permission). (B) Without expression of scn8a (Na_V1.6), Na currents from Purkinje cells show faster inactivation and smaller resurgent currents. Sample currents from a wild-type (black) and null mutant (red) Purkinje cell ((76), with permission). (C) Resurgent current is restored in Purkinje cells lacking scn8a expression by slowing fast inactivation with the site-3 toxin β-pompilidotoxin (β-PMTX). Na currents from an scn8a-null (med) Purkinje cells before and after application of the toxin. Note the slowing of transient current inactivation as well as the appearance of resurgent current ((75), with permission, copyright 2004, Society for Neuroscience). (D) Slowing inactivation with the site-3 toxin ATX-II lowers the affinity of the putative endogenous blocker. Resurgent current evoked upon repolarization to −30 mV averaged across several Purkinje cells and normalized to show the briefer rise time in the presence of toxin ((69), with permission).

Figure 5

Permeation-dependence of unblocking and the resulting resurgent current. (A) Schematic of an open, blocked Na channel α subunit (left) and expulsion of the blocking particle by inward flux of Na⁺ ions (right). Labels: Sans-serif Roman numerals I–IV, the four domains; I with serifs, the fast-inactivation domain; B, a blocking particle ((84), with permission). (B) Resurgent current is disproportionately increased by raising external Na⁺ ions. Left: transient and resurgent currents recorded from a neuron isolated from the mouse cerebellar nuclei (CbN), in 50 mM Na (low Na/Ca) and 155 mM Na (high Na/Ca). The transient current at +30 mV increases 4.5-fold and the resurgent current at −30 mV increases 8-fold, despite a lower proportionate contribution of the change in driving force. Inset shows the resurgent current at higher gain. Right: current-voltage relation for resurgent current in low and high Na ((85), with permission). (C) Resurgent current depends on inward flux of Na ions. Left: simulations from a Markov model with resurgent current generated by permeation-dependent open-channel block and unblock (colored traces). Right: experimental currents recorded from different Purkinje cells with gradients as indicated (black). The same voltage protocol pertains to all sets of traces. Lower families of traces are horizontally offset for clarity, and upward and downward arrows indicate the onset and offset of the 10-ms depolarization to +30 mV. Resurgent current is evident upon repolarization from +30 mV regardless of the direction of the preceding transient current, but outward resurgent current is always very small or undetectable, even with the strongest reverse gradient (inset). Note similarity between Na currents in the near-symmetrical gradient and Shaker K currents with a similar gradient in Fig. 3B (data from (37), with permission, copyright 2010, Society for Neuroscience; simulations from (84), with permission).

Figure 6

Candidate proteins involved in resurgent current in different cells. (A) The cytoplasmic tail of Na_Vβ4 (scn4b) reproduces the magnitude and kinetics of resurgent Na current in Purkinje cells. Representative trace of transient currents evoked by steps from −90 to 0 mV (left) and resurgent currents evoked by steps to −30 mV after 10 ms at +30 mV (right) in control conditions (black), after proteolytic cleavage of the putative blocker (gray), and after application of 200 μM of the β4 peptide (red) ((70), with permission). (B) Deletion of scn4b reduces resurgent current in medium spiny neurons. Sample traces from neurons from wild-type (left) and knockout (right) mice ((116), with permission). (C) Knockdown of Na_Vβ4 reduces resurgent current in cultured cerebellar granule cells. Sample traces of resurgent currents in three representative cells, transfected with either control non-targeted siRNA (black), on-target siRNA to knock down Na_Vβ4 expression (red), or on-target siRNA with the β4 peptide added to the intracellular solution (blue) ((117), with permission) (D) Expression of wild-type but not mutant Na_Vβ4 induces resurgent current in expressed Na_V1.6 channels engineered to be TTX-resistant (Na_V1.6_r) in dorsal root ganglion neurons. Left: schematic of Na_Vβ4 with the lysine residues expected to be sensitive to knockoff highlighted in the wild-type and substituted with alanine residues in the mutant. Right: A representative family of negligible currents (gray) evoked by repolarization with co-transfection of DRG cells with Na_V1.6_r and the alanine mutant Na_Vβ4, with the largest current in red, with the corresponding resurgent currents from Na_V1.6_r alone (black) or co-transfected with wild-type Na_Vβ4 (blue) ((118), with permission). (E) Expression of FGF14-1a (FHF4A) induces resurgent current in expressed Na_V1.8 channels but not Na_V1.6 channels. Representative families of Na currents evoked by repolarization following brief depolarizations in Na_V1.8 alone (left) or with co-transfected FGF14-1a (FHF4A) (middle), illustrating the first successful reconstitution of resurgent Na current in an expression system. Right: expressed Na_V1.6 generates no resurgent current with or without co-transfection of FGF14-1a (FHF4A) or either the A or B isoform of the related protein FGF13 (FHF2) ((109), with permission).