Open-channel block by the cytoplasmic tail of sodium channel beta4 as a mechanism for resurgent sodium current

Voltage-gated sodium channels with "resurgent" kinetics are specialized for high-frequency firing. The alpha subunits interact with a blocking protein that binds open channels upon depolarization and unbinds upon repolarization, producing resurgent sodium current. By limiting classical inactivation, the cycle of block and unblock shortens refractory periods. To characterize the blocker in Purkinje neurons, we briefly exposed inside-out patches to substrate-specific proteases. Trypsin and chymotrypsin each removed resurgent current, consistent with established roles for positively charged and hydrophobic/aromatic groups in blocking sodium channels. In Purkinje cells, the only known sodium channel-associated subunit that has a cytoplasmic sequence with several positive charges and clustered hydrophobic/aromatic residues is beta4 (KKLITFILKKTREK; beta4(154-167)). After enzymatic removal of block, beta4(154-167) fully reconstituted resurgent current, whereas scrambled or point-mutated peptides were ineffective. In CA3 pyramidal neurons, which lack beta4 and endogenous block, beta4(154-167) generated resurgent current. Thus, beta4 may be the endogenous open-channel blocker responsible for resurgent kinetics.     

Inactivation and recovery of sodium currents in cerebellar Purkinje neurons: evidence for two mechanisms

We examined the kinetics of voltage-dependent sodium currents in cerebellar Purkinje neurons using whole-cell recording from dissociated neurons. Unlike sodium currents in other cells, recovery from inactivation in Purkinje neurons is accompanied by a sizeable ionic current. Additionally, the extent and speed of recovery depend markedly on the voltage and duration of the prepulse that produces inactivation. Recovery is faster after brief, large depolarizations (e.g., 5 ms at +30 mV) than after long, smaller depolarizations (e.g., 100 ms at -30 mV). On repolarization to -40 mV following brief, large depolarizations, a resurgent sodium current rises and decays in parallel with partial, nonmonotonic recovery from inactivation. These phenomena can be explained by a model that incorporates two mechanisms of inactivation: a conventional mechanism, from which channels recover without conducting current, and a second mechanism, favored by brief, large depolarizations, from which channels recover by passing transiently through the open state. The second mechanism is consistent with voltage-dependent block of channels by a particle that can enter and exit only when channels are open. The sodium current flowing during recovery from this blocked state may depolarize cells immediately after an action potential, promoting the high-frequency firing typical of Purkinje neurons.     

Nav1.6 channels generate resurgent sodium currents in spinal sensory neurons

The Na(v)1.6 voltage-gated sodium channel has been implicated in the generation of resurgent currents in cerebellar Purkinje neurons. Our data show that resurgent sodium currents are produced by some large diameter dorsal root ganglion (DRG) neurons from wild-type mice, but not from Na(v)1.6-null mice; small DRG neurons do not produce resurgent currents. Many, but not all, DRG neurons transfected with Na(v)1.6 produce resurgent currents. These results demonstrate for the first time the intrinsic ability of Na(v)1.6 to produce a resurgent current, and also show that cell background is critical in permitting the generation of these currents.     

Resurgent Na+ current: a new avenue to neuronal excitability control

Integrative and firing properties are important characteristics of neuronal circuits and these responses are determined in large part by the repertoire of ion channels they express, which can vary considerably between cell types. Recently, a new mode of operation of voltage dependent sodium channels has been described that generates a so-called resurgent Na+ current. Accumulating evidence suggests resurgent Na current participates in the generation of sub-threshold inward Na+ current causing membrane depolarization which provides the necessary drive to fire high-frequency action potentials. Recent studies indicate that resurgent Na+ current could be a more widespread feature than previously thought.     

Inherited Pain: SODIUM CHANNEL NAV1.7 A1632T MUTATION CAUSES ERYTHROMELALGIA DUE TO A SHIFT OF FAST INACTIVATION*

Inherited erythromelalgia (IEM) causes debilitating episodic neuropathic pain characterized by burning in the extremities. Inherited “paroxysmal extreme pain disorder” (PEPD) differs in its clinical picture and affects proximal body areas like the rectal, ocular, or jaw regions. Both pain syndromes have been linked to mutations in the voltage-gated sodium channel Nav1.7. Electrophysiological characterization shows that IEM-causing mutations generally enhance activation, whereas mutations leading to PEPD alter fast inactivation. Previously, an A1632E mutation of a patient with overlapping symptoms of IEM and PEPD was reported (Estacion, M., Dib-Hajj, S. D., Benke, P. J., Te Morsche, R. H., Eastman, E. M., Macala, L. J., Drenth, J. P., and Waxman, S. G. (2008) NaV1.7 Gain-of-function mutations as a continuum. A1632E displays physiological changes associated with erythromelalgia and paroxysmal extreme pain disorder mutations and produces symptoms of both disorders. J. Neurosci. 28, 11079–11088), displaying a shift of both activation and fast inactivation. Here, we characterize a new mutation of Nav1.7, A1632T, found in a patient suffering from IEM. Although transfection of A1632T in sensory neurons resulted in hyperexcitability and spontaneous firing of dorsal root ganglia (DRG) neurons, whole-cell patch clamp of transfected HEK cells revealed that Nav1.7 activation was unaltered by the A1632T mutation but that steady-state fast inactivation was shifted to more depolarized potentials. This is a characteristic normally attributed to PEPD-causing mutations. In contrast to the IEM/PEPD crossover mutation A1632E, A1632T failed to slow current decay (i.e. open-state inactivation) and did not increase resurgent currents, which have been suggested to contribute to high-frequency firing in physiological and pathological conditions. Reduced fast inactivation without increased resurgent currents induces symptoms of IEM, not PEPD, in the new Nav1.7 mutation, A1632T. Therefore, persistent and resurgent currents are likely to determine whether a mutation in Nav1.7 leads to IEM or PEPD.     

Interactions among DIV voltage-sensor movement,fast inactivation,and resurgent Na current induced by the NaVβ4 open-channel blocking peptide

Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the Na_Vβ4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed Na_V1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of Na_Vβ4 (the “β4 peptide”). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; “CW” channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the β4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein.     

Simulation of Na channel inactivation by thiazine dyes

Some dyes of the methylene blue family serve as artificial inactivators of the sodium channels when present inside squid axons at a concentration of approximately 0.1 mM. The dyes restore a semblance of inactivation after normal inactivation has been destroyed by pronase. In fibers that inactivate normally, the dyes hasten the decay of sodium current. Many dye-blocked channels conduct transiently on exit of the dye molecule after repolarization to the holding potential. In contrast, normally inactivated channels do not conduct during recovery from inactivation. Kinetic evidence shows that inactivation of a dye-blocked channel is unlikely or impossible, which suggests that dye molecules compete with inactivation "particles" for the same site. In the absence of tetrodotoxin, the dyes do not affect the ON gating current unless the interpulse interval is very short. If sufficient equilibration time is allowed during a pulse, the initial amplitude of the OFF gating current is reduced to near zero. This suggests that a dye molecule is a Na channel completely blocks that channel's gating current, even the fraction that is resistant to normal inactivation. Dyes block INa and Ig with the same time course. This provides the strongest evidence to date that virtually all of recorded "gating current" is associated with Na channels. Tetrodotoxin greatly slows dissociation of dye molecules from Na channels and reduced gating current during both opening and closing of the channels.     

Protein kinase C enhances human sodium channel hNav1.7 resurgent currents via a serine residue in the domain III–IV linker

Resurgent sodium currents likely play a role in modulating neuronal excitability. Here we studied whether protein kinase C (PKC) activation can increase resurgent currents produced by the human sodium channel hNav1.7. We found that a PKC agonist significantly enhanced hNav1.7-mediated resurgent currents and this was prevented by PKC antagonists. The enhancing effects were replicated by two phosphorylation-mimicking mutations and were prevented by a phosphorylation-deficient mutation at a conserved PKC phosphorylation site (Serine 1479). Our results suggest that PKC can increase sodium resurgent currents through phosphorylation of a conserved Serine residue located in the domain III–IV linker of sodium channels.     

Resurgent current and voltage sensor trapping enhanced activation by a beta-scorpion toxin solely in Nav1.6 channel. Significance in mice Purkinje neurons

Resurgent currents are functionally crucial in sustaining the high frequency firing of cerebellar Purkinje neurons expressing Na(v)1.6 channels. Beta-scorpion toxins, such as CssIV, induce a left shift in the voltage-dependent activation of Na(v)1.2 channels by "trapping" the IIS4 voltage sensor segment. We found that the dangerous Cn2 beta-scorpion peptide induces both the left shift voltage-dependent activation and a transient resurgent current only in human Na(v)1.6 channels (among 1.1-1.7), whereas CssIV did not induce the resurgent current. Cn2 also produced both actions in mouse Purkinje cells. These findings suggest that only distinct beta-toxins produce resurgent currents. We suggest that the novel and unique selectivity of Cn2 could make it a model drug to replace deep brain stimulation of the subthalamic nucleus in patients with Parkinson disease.     

Subthreshold sodium current from rapidly inactivating sodium channels drives spontaneous firing of tuberomammillary neurons

A role for "persistent," subthreshold, TTX-sensitive sodium current in driving the pacemaking of many central neurons has been proposed, but this has been impossible to test pharmacologically. Using isolated tuberomammillary neurons, we assessed the role of subthreshold sodium current in pacemaking by performing voltage-clamp experiments using a cell's own pacemaking cycle as voltage command. TTX-sensitive sodium current flows throughout the pacemaking cycle, even at voltages as negative as -70 mV, and this current is sufficient to drive spontaneous firing. When sodium channels underlying transient current were driven into slow inactivation by rapid stimulation, persistent current decreased in parallel, suggesting that persistent sodium current originates from subthreshold gating of the same sodium channels that underlie the phasic sodium current. This behavior of sodium channels may endow all neurons with an intrinsic propensity for rhythmic, spontaneous firing.     

Ultra-slow inactivation of the ionic currents through the membrane of myelinated nerve.

(1) Voltage-clamp experiments were performed with myelinated fibres isolated from the sciatic nerve of the frog to study slow changes of the specific sodium and potassium currents as a function of membrane (holding) potential and time. (2) The level of the peak sodium current depends on holding potential VH. This dependence can be described by a sigmoidal function uinfinity(VH). The underlying process is called "ultra-slow sodium inactivation" and is different and separable from the short time steady-state inactivation, hinfinity(V), and from the slow inactivation depending on the extracellular potassium concentration (Adelman, Jr., W. J. and Palti, Y. (1969), J Gen. Physiol. 54, 589-606; Peganov, E. M., Khodorov, B.I. and Shishkova, L. D. (1973), Bull. Exp. Biol. Med. 25, 15-19; Khodorov, B. I. Shishkova, L. D. and Peganov, E. M. (1974), Bull. Exp. Biol. Med. 3, 10-14). (3) After a sudden change of the holding potential the sodium current reaches a new steady-state level (due to the transition of uinfinity(VH) to the corresponding value) within approx. 4 min. The kinetics of the transition cannot be described by a single exponential function. (4) A corresponding voltage- and time-dependent process of ultra-slow inactivation exists for the potassium current in the node of Ranvier. The kinetics are faster than those of the sodium system.     

Gating of the squid sodium channel at positive potentials. I. Macroscopic ionic and gating currents.

Macroscopic ionic sodium currents and gating currents were studied in voltage-clamped, dialyzed giant axons of the squid Loligo pealei under conditions of regular and inverse sodium gradients. Sodium currents showed regular kinetics but inactivation was incomplete, showing a maintained current for depolarizations lasting 18 ms. The ratio of the maintained current to the peak current increased with depolarization and it did not depend on the direction of the current flow or the sodium gradient. The time constant of inactivation was not affected by the sodium gradient. Double-pulse experiments allowed the separation of a normal inactivating component and a noninactivating component of the sodium currents. In gating current experiments, the results from double-pulse protocols showed that the charge was decreased by the prepulse and that the slow component of the 'on' gating current was preferentially depressed. As expected, charge immobilization was established faster at higher depolarizations than at low depolarizations, however, the amount of immobilized charge was unaffected by the pulse amplitude. This indicates that the incomplete sodium inactivation observed at high depolarizations is not the result of decreased charge immobilization; the maintained current must be due to a conductance that appears after normal charge immobilization and fast inactivation.     

Gating current "fractionation" in crayfish giant axons.

Effects of changes in initial conditions on the magnitude and kinetics of gating current and sodium current were studied in voltage-clamped, internally-perfused, crayfish giant axons. We examined the effects of changes in holding potential, inactivating prepulses, and recovery from inactivation in axons with intact fast inactivation. We also studied the effects of brief interpulse intervals in axons pretreated with chloramine-T for removal of fast inactivation. We find marked effects of gating current kinetics induced by both prepulse inactivation and brief interpulse intervals. The apparent changes in gating current relaxation rates cannot be explained simply by changes in gating charge magnitude (charge immobilization) combined with "Cole-Moore-type" time shifts. Rather they appear to indicate selective suppression of kinetically-identifiable components within the control gating currents. Our results provide additional support for a model involving parallel, nonidentical, gating particles.     

Gating current harmonics. IV. Dynamic properties of secondary activation kinetics in sodium channel gating.

The kinetics for sodium channel gating appear to involve three coupled processes: (a) "primary" activation, (b) "secondary" activation, and (c) inactivation. Gating current data obtained in dynamic steady states with sinusoidal voltage-clamp were analyzed to give further details about the secondary activation process in sodium channel gating. Unlike primary activation and inactivation, the secondary activation kinetics involve physical processes that become defined when the data are analyzed as a function of the sinusoid frequency in addition to mean membrane potential. The effects of these processes are described, and a physical interpretation is presented.     

Interaction of n-alkylguanidines with the sodium channels of squid axon membrane

The effects of n-alkylguanidine derivatives on sodium channel conductance were measured in voltage clamped, internally perfused squid giant axons. After destruction of the sodium inactivation mechanism by internal pronase treatment, internal application of n-amylguanidine (0.5 mM) or n-octylguanidine (0.03 mM) caused a time-dependent block of sodium channels. No time-dependent block was observed with shorter chain derivatives. No change in the rising phase of sodium current was seen and the block of steady-state sodium current was independent of the membrane potential. In axons with intact sodium inactivation, an apparent facilitation of inactivation was observed after application of either n-amylguanidine or n-octylguanidine. These results can be explained by a model in which alkylguanidines enter and occlude open sodium channels from inside the membrane with voltage-independent rate constants. Alkylguanidine block bears a close resemblance to natural sodium inactivation. This might be explained by the fact that alkylguanidines are related to arginine, which has a guanidino group and is thought to be an essential amino acid in the molecular mechanism of sodium inactivation. A strong correlation between alkyl chain length and blocking potency was found, suggesting that a hydrophobic binding site exists near the inner mouth of the sodium channel.     

Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation

Sodium-calcium exchange current was isolated in inside-out patches excised from guinea pig ventricular cells using the giant patch method. The outward exchange current decayed exponentially upon activation by cytoplasmic sodium (sodium-dependent inactivation). The kinetics and mechanism of the inactivation were studied. (a) The rate of inactivation and the peak current amplitude were both strongly temperature dependent (Q10 = 2.2). (b) An increase in cytoplasmic pH from 6.8 to 7.8 attenuated the current decay and shifted the apparent dissociation constant (Kd) of cytoplasmic calcium for secondary activation of the exchange current from 9.6 microM to < 0.3 microM. (c) The amplitude of exchange current decreased synchronously over the membrane potential range from -120 to 60 mV during the inactivation, indicating that voltage dependence of the exchanger did not change during the inactivation process. The voltage dependence of exchange current also did not change during secondary modulation by cytoplasmic calcium and activation by chymotrypsin. (d) In the presence of 150 mM extracellular sodium and 2 mM extracellular calcium, outward exchange current decayed similarly upon application of cytoplasmic sodium. Upon removal of cytoplasmic sodium in the presence of 2-5 microM cytoplasmic free calcium, the inward exchange current developed in two phases, a fast phase within the time course of solution changes, and a slow phase (tau approximately 4 s) indicative of recovery from sodium-dependent inactivation. (e) Under zero-trans conditions, the inward current was fully activated within solution switch times upon application of cytoplasmic calcium and did not decay. (f) The slow recovery phase of inward current upon removal of cytoplasmic sodium was also present under the zero-trans condition. (g) Sodium-dependent inactivation shows little or no dependence on membrane potential in guinea pig myocyte sarcolemma. (h) Sodium-dependent inactivation of outward current is attenuated in rate and extent as extracellular calcium is decreased. (i) Kinetics of the sodium-dependent inactivation and its dependence on major experimental variables are well described by a simple two-state inactivation model assuming one fully active and one fully inactive exchanger state, whereby the transition to the inactive state takes place from a fully sodium-loaded exchanger conformation with cytoplasmic orientation of binding sites (E1.3Ni).     

Kinetics of the sodium current through EDTA-modified calcium channels of the molluscan neuron somatic membrane

The kinetics of the slow current carried by sodium ions through potential-dependent calcium channels after addition of EDTA to calcium-free external solution was investigated in experiments by the intracellular dialysis method on isolatedHelix pomatia neurons. The activation kinetics of this current was similar to that of the calcium current and could be described by the use of the square of the activation variable m in Hodgkin-Huxley equations. The decay (inactivation) kinetics of the induced sodium current during prolonged depolarization is biexponential in character. It is suggested that decay of the sodium currents takes place as a result of two independent processes: potential-dependent inactivation with a time constant τ_h～1 sec, taking place as far as a certain steady-state level h_∞, and a decrease in current connected with Na⁺ accumulation inside the cell during passage of the current and a consequent change in the sodium electrochemical potential (τ_c～10 sec). It is concluded that modification of the calcium channels, so that they acquire the ability to conduct sodium, has no significant effect on the gating mechanisms responsible for opening and closing of the channels.     

Enhancement of closed-state inactivation in long QT syndrome sodium channel mutation DeltaKPQ

DeltaKPQ, a three amino acid [lysine (K), proline (P), glutamine (Q)] deletion mutation of the human cardiac Na channel (hH1), which is one cause of long QT syndrome (LQT3), has impaired inactivation resulting in a late sodium current. To better understand inactivation in DeltaKPQ, we applied a site-3 toxin anthopleurin A, which has been shown to inhibit inactivation from the open state with little or no effect on inactivation from the closed state(s) in wild-type hH1. In contrast to the effect of site-3 toxins on wild-type hH1, inactivation from closed state(s) in toxin-modified DeltaKPQ demonstrated a large negative shift in the Na channel availability curve of nearly -14 mV. Recovery from inactivation showed that toxin-modified DeltaKPQ channels recovered slightly faster than those in control, whereas development of inactivation at potentials negative to -80 mV showed that inactivation developed much more rapidly in toxin-modified DeltaKPQ channels compared with control. An explanation for our results is that closed-state inactivation in toxin-modified DeltaKPQ is enhanced by the mutated inactivation lid being positioned "closer" to its receptor resulting in an increased rate of association between the inactivation lid and its receptor.     

A slow component of gating current in crayfish giant axons resembles inactivation charge movement.

Recent experimental evidence from a number of preparations indicates that sodium channel inactivation may be intrinsically voltage sensitive. Intrinsically voltage sensitive inactivation should produce a charge movement. Crayfish giant axons provide a unique opportunity to reexamine the slower components of gating currents (Ig) for a contribution from inactivation (Igh). In reference to other axon preparations, this preparation has relatively rapid inactivation, and steady-state inactivation has a comparatively steep voltage dependence. As predicted by a two-state scheme for voltage-sensitive sodium channel inactivation, Ig in crayfish axons includes a slow component with time constant comparable to the time constant of decay of the sodium current. Allowing for some delay in its onset (60 microseconds), inactivation as described by this slow component of Ig carries roughly the amount of charge predicted by the voltage dependence of inactivation.