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Functional analysis of disease-associated mutant NaV1.4 channels have revealed defects that often provide a mechanistic basis for derangements in sarcolemma excitability to produce the clinical phenotype. Loss-of-function mutations are less common and the pathogenic basis for muscle dysfunction is less clear than for the gain-of-function NaV1.4 mutations associated with myotonia, paramyotonia congenita or hyperkalemic periodic paralysis. Loss-of-function via enhanced inactivation (both fast and slow) accompanies other channel defects in hypokalemic periodic paralysis (HypoKPP) and is the primary defect in a rare form of congenital myasthenic syndrome. We explored whether a NaV1.4 loss-of-function created in a mouse would produce features of HypoKPP or myasthenia.A null allele of NaV1.4 was generated by excision of exon 12 (174 bp which encodes DIIS2-DIIS4). Heterozygous (+/-) mice were viable, but homozygous nulls (-/-) did not survive beyond P2. Spontaneous movement, feeding, and reproduction were normal in (+/-) mice. Voltage-clamp studies of dissociated fibers showed a 50% reduction of Ina in (+/-) animals. The Na current was completely blocked by 200 nM TTX, suggesting there was no compensatory upregulation of NaV1.5. In vitro contraction testing showed a right shift of the force- stimulus intensity curve and a prominent sag from the early peak force for submaximal stimulus intensities in (+/-) mice. Repetitive nerve stimulation did not elicit a decremental response in the compound muscle AP (CMAP) at baseline, but exposure to low-dose curare (0.5 uM) revealed a larger decrement for (+/-) than WT. These features are consistent with mild myasthenia. Challenge with low K did not diminish maximal tetanic force, nor did insulin & glucose infusion cause a decrease of CMAP amplitude, both of which were observed in our R669H HypoKPP mice.

Muscle fibers from individuals with hyperkalemic periodic paralysis generate repetitive trains of action potentials (myotonia) or large depolarizations and block of spike production (paralysis) when the extracellular K+ is elevated. These pathologic features are thought to arise from mutations of the sodium channel alpha subunit which cause a partial loss of inactivation (steady-state Popen approximately 0.02, compared to < 0.001 in normal channels). We present a model that provides a possible mechanism for how this small persistent sodium current leads to repetitive firing, why the integrity of the T-tubule system is required to produce myotonia, and why paralysis will occur when a slightly larger proportion of channels fails to inactivate. The model consists of a two-compartment system to simulate the surface and T-tubule membranes. When the steady-state sodium channel open probability exceeds 0.0075, trains of repetitive discharges occur in response to constant current injection. At the end of the current injection, the membrane potential may either return to the normal resting value, continue to discharge repetitive spikes, or settle to a new depolarized equilibrium potential. This after-response depends on both the proportion of noninactivating sodium channels and the magnitude of the activity-driven K+ accumulation in the T-tubular space. A reduced form of model is presented in which a two-dimensional phase-plane analysis shows graphically how this diversity of after-responses arises as extracellular [K+] and the proportion of noninactivating sodium channels are varied.

Mutations in the coding sequence of voltage-gated ionic channels are known to cause a wide variety of diseases affecting muscle and brain. Biophysical studies on the functional consequences of these defects are now revealing an equally diverse spectrum of mechanisms that underlie the disruption of cellular excitability, synaptic transmission, or neuronal survival. This Workshop on Channelopathies highlights recent advances in understanding the mechanistic connection between altered channel behavior and disease pathogenesis. New knock-in mouse models of Familial Hemiplegic Migraine illustrate how subtle gain-of-function changes in P/Q-type CaV2.1 channels enhance excitatory synaptic transmission and promote cortical spreading depression. A transmembrane protein linked to Familial Alzheimer Disease (presenilin) has recently been shown to form an unconventional Ca2+ leak channel that accounts for 80% of the divalent conductance of the ER. Finally, new insights have emerged in the past two years on a possible common pathomechanism by which mutations in either NaV1.4 or CaV1.1 channels of skeletal muscle may cause periodic paralysis. In both cases, mutations are clustered at arginine residues of the S4 voltage-sensor domain. Mutant channels conduct small “omega” currents through a voltage-regulated gating pore and may be the source of the inward current that renders affected fibers susceptible to sustained depolarized shifts during attacks of weakness.

The mechanism whereby missense mutations in charged residues of the S4 segments of CaV1.1 and NaV1.4 cause the skeletal muscle disorder hypokalemic periodic paralysis (HypoPP) remains poorly understood. Recent work suggests a possible common functional defect, in which HypoPP mutations produce aberrant ionic conductances flowing through the aqueous gating-pore in which the mutant S4 segment resides. We observed low-amplitude gating-pore currents for HypoPP mutations in the R1 and R2 positions of S4 in domain II in NaV1.4. Several features of these HypoPP-associated gating pore conductances were unexpected, and may provide insight into S4 segment function. For instance, gating pores exposed by mutations at the R2 site exhibited marked current saturation at hyperpolarized voltages. Saturation can be accounted for by a model with a single cation binding site very near the external surface of the electrical field. The ionic selectivity of different HypoPP gating pores is dependent on the substituted residue: histidine substitutions causing proton-selectivity, whereas other substitutions result in limited selectivity among monovalent cations. The pathophysiological significance of this dichotomy remains unclear. In addition, the low amplitude of the disease-associated gating pore currents (∼.1% of the peak Na current through the central pore) is probably insufficient to directly cause the large depolarization of affected muscle fibers during a paralytic attack. These small currents might predispose to episodic paralysis by potentiating the normal sarcolemmal propensity to depolarize upon reduction of external K+. This paradoxical depolarization is a consequence of the K+ dependence for the inward rectifier K+ conductance, which causes Vrest to deviate from Nernstian behavior. Thus, muscle fibers with an inward gating-pore current may function normally at most times, but may be poised for massive depolarization in the setting of minor perturbations of extracellular [K+].

All eight missense mutations of NaV1.4 associated with hypokalemic periodic paralysis (HypoPP) occur at arginine residues in S4 voltage sensors. These R/X mutations create an anomalous ion conduction pathway, detected as a gating pore current (Igp) in the oocyte. A critical question is how large is the gating pore conductance in HypoPP muscle, since this will determine susceptibility to depolarization-induced weakness. Large discrepancies for estimates of Igp relative to peak INa have been reported; Igp / INa = ∼ 1:100 when currents are measured with TTX (Igp) and without (INa) in the same egg, whereas the conductance ratio γgp / γNa = ∼ 1:1000 based on measurement of Igp and maximal “on” gating charge displacement, Qon.To resolve this issue, we measured Qon, INa, and Igp in oocytes expressing WT or R672G channels. Robust gating pore currents were observed for R672G channels, with Igp/Qon = 65.2 nA/nC at −140 mV. The ratio of INa(-10 mv) to Qon was decreased 2.3-fold for R672G channels compared to WT. This decoupling may also account for the 2x reduction of INa in muscle fibers from R669H knock-in HypoPP mouse for which mRNA levels were not reduced. For R672G, the measured currents had a ratio of Igp(-140) / INa(-10) = 0.025 or 1:40. This current ratio can be converted to an equivalent conductance ratio by accounting for (i) decoupling 2.3, (ii) Popen_max = 0.25, (iii) driving force of 140 mV for Igp and 50 mV for INa. All together, these factors are ∼25 and yield an equivalent conductance ratio of 1: 40 x 25 = 1 : 1000; thereby resolving the discrepancy in the literature.Supported by NIAMS and MDA.

Mutations in segment IVS6 of voltage-gated Na + channels affect fast-inactivation, slow-inactivation, local anesthetic action, and batrachotoxin (BTX) action. To detect conformational changes associated with these processes, we substituted a cysteine for a valine at position 1583 in the rat adult skeletal muscle sodium channel α -subunit, and examined the accessibility of the substituted cysteine to modification by 2-aminoethyl methanethiosulfonate (MTS-EA) in excised macropatches. MTS-EA causes an irreversible reduction in the peak current when applied both internally and externally, with a reaction rate that is strongly voltage-dependent. The rate increased when exposures to MTS-EA occurred during brief conditioning pulses to progressively more depolarized voltages, but decreased when exposures occurred at the end of prolonged depolarizations, revealing two conformational changes near site 1583, one coupled to fast inactivation, and one tightly associated with slow inactivation. Tetraethylammonium, a pore blocker, did not affect the reaction rate from either direction, while BTX, a lipophilic activator of sodium channels, completely prevented the modification reaction from occurring from either direction. We conclude that there are two inactivation-associated conformational changes in the vicinity of site 1583, that the reactive site most likely faces away from the pore, and that site 1583 comprises part of the BTX receptor.

Over 20 different missense mutations in the alpha subunit of the adult skeletal muscle Na channel have been identified in families with either myotonia (muscle stiffness) or periodic paralysis, or both. The V445M mutation was recently found in a family with myotonia but no weakness. This mutation in transmembrane segment IS6 is novel because no other disease-associated mutations are in domain I. Na currents were recorded from V445M and wild-type channels transiently expressed in human embryonic kidney cells. In common with other myotonic mutants studied to date, fast gating behavior was altered by V445M in a manner predicted to increase excitability: an impairment of fast inactivation increased the persistent Na current at 10 ms and activation had a hyperpolarized shift (4 mV). In contrast, slow inactivation was enhanced by V445M due to both a slower recovery (10 mV left shift in beta(V)) and an accelerated entry rate (1.6-fold). Our results provide additional evidence that IS6 is crucial for slow inactivation and show that enhanced slow inactivation cannot prevent myotonia, whereas previous studies have shown that disrupted slow inactivation predisposes to episodic paralysis.