Peter C. Ruben

Voltage Sensors in Domains III and IV, but Not I and II, Are Immobilized by Na+ Channel Fast Inactivation

Using site-directed fluorescent labeling, we examined conformational changes in the S4 segment of each domain of the human skeletal muscle sodium channel (hSkM1). The fluorescence signals from S4 segments in domains I and II follow activation and are unaffected as fast inactivation settles. In contrast, the fluorescence signals from S4 segments in domains III and IV show kinetic components during activation and deactivation that correlate with fast inactivation and charge immobilization. These results indicate that in hSkM1, the S4 segments in domains III and IV are responsible for voltage-sensitive conformational changes linked to fast inactivation and are immobilized by fast inactivation, while the S4 segments in domains I and II are unaffected by fast inactivation.

Evolutionary diversification of TTX-resistant sodium channels in a predator–prey interaction

Understanding the molecular genetic basis of adaptations provides incomparable insight into the genetic mechanisms by which evolutionary diversification takes place. Whether the evolution of common traits in different lineages proceeds by similar or unique mutations, and the degree to which phenotypic evolution is controlled by changes in gene regulation as opposed to gene function, are fundamental questions in evolutionary biology that require such an understanding of genetic mechanisms. Here we identify novel changes in the molecular structure of a sodium channel expressed in snake skeletal muscle, tsNaV1.4, that are responsible for differences in tetrodotoxin (TTX) resistance among garter snake populations coevolving with toxic newts. By the functional expression of tsNaV1.4, we show how differences in the amino-acid sequence of the channel affect TTX binding and impart different levels of resistance in four snake populations. These results indicate that the evolution of a physiological trait has occurred through a series of unique functional changes in a gene that is otherwise highly conserved among vertebrates.

Slow inactivation in voltage-gated sodium channels

Slow inactivation in voltage-gated sodium channels is a biophysical process that governs the availability of sodium channels over extended periods of time. Slow inactivation, therefore, plays an important role in controlling membrane excitability, firing properties, and spike frequency adaptation. Defective slow inactivation is associated with several diseases of cell excitability, such as hyperkalemic periodic paralysis, myotonia, idiopathic ventricular fibrillation and long-QT syndrome. These associations underscore the physiological importance of this phenomenon. Nevertheless, our understanding of the molecular substrates for slow inactivation is still fragmentary. This review covers the current state of knowledge concerning the molecular underpinnings of slow inactivation, and its relationship with other biophysical processes of voltage-gated sodium channels.

Exome Sequencing and the Management of Neurometabolic Disorders

BACKGROUND Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patients clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Childrens Hospital Foundation and others.).

Slow Inactivation in Human Cardiac Sodium Channels

The available pool of sodium channels, and thus cell excitability, is regulated by both fast and slow inactivation. In cardiac tissue, the requirement for sustained firing of long-duration action potentials suggests that slow inactivation in cardiac sodium channels may differ from slow inactivation in skeletal muscle sodium channels. To test this hypothesis, we used the macropatch technique to characterize slow inactivation in human cardiac sodium channels heterologously expressed in Xenopus oocytes. Slow inactivation was isolated from fast inactivation kinetically (by selectively recovering channels from fast inactivation before measurement of slow inactivation) and structurally (by modification of fast inactivation by mutation of IFM1488QQQ). Time constants of slow inactivation in cardiac sodium channels were larger than previously reported for skeletal muscle sodium channels. In addition, steady-state slow inactivation was only 40% complete in cardiac sodium channels, compared to 80% in skeletal muscle channels. These results suggest that cardiac sodium channel slow inactivation is adapted for the sustained depolarizations found in normally functioning cardiac tissue. Complete slow inactivation in the fast inactivation modified IFM1488QQQ cardiac channel mutant suggests that this impairment of slow inactivation may result from an interaction between fast and slow inactivation.

Interaction between voltage-gated sodium channels and the neurotoxin, tetrodotoxin

Tetrodotoxin (TTX) is a potent toxin that specifically binds to voltage gated sodium channels. TTX binding physically blocks the flow of sodium ions through the channel, thereby preventing action potential (AP) generation and propagation. TTX has different binding affinities for different sodium channel isoforms. These differences are imparted by amino acid substitutions. Such substitutions confer TTX resistance to a variety of species. Tetrodotoxin resistance, however, may come at a cost to performance caused by changes in the biophysical properties and/or ion selectivity of the TTX resistant sodium channels. We here review the properties of sodium channels and their interaction with TTX, and look at some special examples of TTX resistant channels wherein the benefit of toxin resistance may be offset by other behavioral costs.

Structural determinants of slow inactivation in human cardiac and skeletal muscle sodium channels.

Skeletal and heart muscle excitability is based upon the pool of available sodium channels as determined by both fast and slow inactivation. Slow inactivation in hH1 sodium channels significantly differs from slow inactivation in hSkM1. The beta(1)-subunit modulates fast inactivation in human skeletal sodium channels (hSkM1) but has little effect on fast inactivation in human cardiac sodium channels (hH1). The role of the beta(1)-subunit in sodium channel slow inactivation is still unknown. We used the macropatch technique on Xenopus oocytes to study hSkM1 and hH1 slow inactivation with and without beta(1)-subunit coexpression. Our results indicate that the beta(1)-subunit is partly responsible for differences in steady-state slow inactivation between hSkM1 and hH1 channels. We also studied a sodium channel chimera, in which P-loops from each domain in hSkM1 sodium channels were replaced with corresponding regions from hH1. Our results show that these chimeras exhibit hH1-like properties of steady-state slow inactivation. These data suggest that P-loops are structural determinants of sodium channel slow inactivation, and that the beta(1)-subunit modulates slow inactivation in hSkM1 but not hH1. Changes in slow inactivation time constants in sodium channels coexpressed with the beta(1)-subunit indicate possible interactions among the beta(1)-subunit, P-loops, and the slow inactivation gate in sodium channels.

A defect in skeletal muscle sodium channel deactivation exacerbates hyperexcitability in human paramyotonia congenita

1 Paramyotonia congenita (PC) is a human hereditary disorder wherein missense mutations in the skeletal muscle sodium channel lead to cold‐exacerbated muscle hyperexcitability. The most common site for PC mutations is the outermost arginine of domain IV segment 4 (human R1448, rat R1441). 2 We examined the rat homologues of two PC mutants with changes at this site: R1441P and R1441C. The R→P mutation leads to the most clinically severe form of the disease. Since PC has so far been attributed to defects in fast inactivation, we expected the R→P substitution to have a more dramatic effect on fast inactivation than R→C. Both mutants (R1441P and R1441C), however, had identical rates and voltage dependence of fast inactivation and activation. 3 R1441P and R1441C also had slowed deactivation, compared with wild‐type, raising the possibility that slowed deactivation, in combination with defective fast inactivation, might be a contributing cause of paramyotonia congenita. Furthermore, deactivation was slower in R1441P than in R1441C, suggesting that the worse phenotype of the human R→P mutation is due to a greater effect on deactivation, and supporting our hypothesis that slowed sodium channel deactivation contributes to paramyotonia congenita. 4 We show that the downstroke of the muscle action potential produced a sodium tail current, and thus slowed deactivation opposes repolarization and therefore leads to hyperexcitability. Hyperexcitability due to slowed deactivation, which has previously been overlooked, also predicts the temperature sensitivity of PC, which has otherwise not been adequately explained.

A Single Residue Differentiates between Human Cardiac and Skeletal Muscle Na+ Channel Slow Inactivation

Slow inactivation determines the availability of voltage-gated sodium channels during prolonged depolarization. Slow inactivation in hNa(V)1.4 channels occurs with a higher probability than hNa(V)1.5 sodium channels; however, the precise molecular mechanism for this difference remains unclear. Using the macropatch technique we show that the DII S5-S6 p-region uniquely confers the probability of slow inactivation from parental hNa(V)1.5 and hNa(V)1.4 channels into chimerical constructs expressed in Xenopus oocytes. Site-directed mutagenesis was used to test whether a specific region within DII S5-S6 controls the probability of slow inactivation. We found that substituting V754 in hNa(V)1.4 with isoleucine from the corresponding position (891) in hNa(V)1.5 produced steady-state slow inactivation statistically indistinguishable from that in wild-type hNa(V)1.5 channels, whereas other mutations have little or no effect on slow inactivation. This result indicates that residues V754 in hNa(V)1.4 and I891in hNa(V)1.5 are unique in determining the probability of slow inactivation characteristic of these isoforms. Exchanging S5-S6 linkers between hNa(V)1.4 and hNa(V)1.5 channels had no consistent effect on the voltage-dependent slow time inactivation constants [tau(V)]. This suggests that the molecular structures regulating rates of entry into and exit from the slow inactivated state are different from those controlling the steady-state probability and reside outside the p-regions.

Defective fast inactivation recovery and deactivation account for sodium channel myotonia in the I1160V mutant

The skeletal muscle sodium channel mutant I1160V cosegregates with a disease phenotype producing myotonic discharges (observed as muscle stiffness) that are worsened by elevated K+ levels but unaffected by cooling. The I1160V alpha-subunit was co-expressed with the beta1-subunit in Xenopus oocytes. An electrophysiological characterization was undertaken to examine the underlying biophysical characteristics imposed by this mutation. Two abnormalities were found. 1) The voltage dependence of steady-state fast inactivation was reduced in I1160V, which resulted in faster rates of closed-state fast inactivation onset and recovery in I1160V compared with wild-type channels. 2) The rates of deactivation were slower in I1160V than in wild-type channels. Using a computer-simulated model, the combination of both defects elicited myotonic runs under conditions of elevated K+, consistent with the observed phenotype of the mutant.