NOS1AP variant associated with incidence of type 2 diabetes in calcium channel blocker users in the Atherosclerosis Risk in Communities (ARIC) study.

AIMS/HYPOTHESIS: To validate the reported association between rs10494366 in NOS1AP (the gene encoding nitric oxide synthase-1 adaptor protein) and the incidence of type 2 diabetes in calcium channel blocker (CCB) users and to identify additional NOS1AP variants associated with type 2 diabetes risk. METHODS: Data from 9 years of follow-up in 9,221 middle-aged white and 2,724 African-American adults free of diabetes at baseline from the Atherosclerosis Risk in Communities study were analysed. Nineteen NOS1AP variants were examined for associations with incident diabetes and fasting glucose levels stratified by baseline CCB use. RESULTS: Prevalence of CCB use at baseline was 2.7% (n = 247) in whites and 2.3% (n = 72) in African-Americans. Among white CCB users, the G allele of rs10494366 was associated with lower diabetes incidence (HR 0.57, 95% CI 0.35-0.92, p = 0.016). The association was marginally significant after adjusting for age, sex, obesity, smoking, alcohol use, physical activity, hypertension, heart rate and electrocardiographic QT interval (HR 0.63, 95% CI 0.38-1.04, p = 0.052). rs10494366 was associated with lower average fasting glucose during follow-up (p = 0.037). No other variants were associated with diabetes risk in CCB users after multiple-testing correction. No associations were observed between any NOS1AP variant and diabetes development in non-CCB users. NOS1AP variants were not associated with diabetes risk in either African-American CCB users or non-CCB users. CONCLUSIONS/INTERPRETATION: We have independently replicated the association between rs10494366 in NOS1AP and incident diabetes among white CCB users. Further exploration of NOS1AP variants and type 2 diabetes and functional studies of NOS1AP in type 2 diabetes pathology is warranted.

Molecular localization of an ion-binding site within the pore of mammalian sodium channels.

Sodium channels are the major proteins that underlie excitability in nerve, heart, and skeletal muscle. Chemical reaction rate theory was used to analyze the blockage of single wild-type and mutant sodium channels by cadmium ions. The affinity of cadmium for the native tetrodotoxin (TTX)-resistant cardiac channel was much higher than its affinity for the TTX-sensitive skeletal muscle isoform of the channel (microliters). Mutation of Tyr401 to Cys, the corresponding residue in the cardiac sequence, rendered microliters highly susceptible to cadmium blockage but resistant to TTX. The binding site was localized approximately 20% of the distance down the electrical field, thus defining the position of a critical residue within the sodium channel pore.

Depth asymmetries of the pore-lining segments of the Na+ channel revealed by cysteine mutagenesis.

We used serial cysteine mutagenesis to study the structure of the outer vestibule and selectivity region of the voltage-gated Na channel. The voltage dependence of Cd(2+) block enabled us to determine the locations within the electrical field of cysteine-substituted mutants in the P segments of all four domains. The fractional electrical distances of the substituted cysteines were compared with the differential sensitivity to modification by sulfhydryl-specific modifying reagents. These experiments indicate that the P segment of domain II is external, while the domain IV P segment is displaced internally, compared with the first and third domain P segments. Sulfhydryls with a steep voltage dependence for Cd(2+) block produced changes in monovalent cation selectivity; these included substitutions at the presumed selectivity filter, as well as residues in the domain IV P segment not previously recognized as determinants of selectivity. A new structural model is presented in which each of the P segments contribute unique loops that penetrate the membrane to varying depths to form the channel pore.

Sodium channels from human brain RNA expressed in Xenopus oocytes. Basic electrophysiologic characteristics and their modification by diphenylhydantoin.

We describe the expression and characterization of sodium channels from human brain RNA in the Xenopus oocyte. The expressed channel, studied by whole-cell voltage clamp, reveals characteristic selectivity for sodium as the permeant ion, voltage-dependent gating, and block by nanomolar concentrations of tetrodotoxin. Such channels are not seen in control oocytes injected with solvent only. The anticonvulsant diphenylhydantoin (DPH) inhibits the expressed channel in a voltage- and use-dependent manner, much like the effect seen in primary mammalian neuronal preparations. The inhibition of the expressed human sodium channel by DPH can be described by models previously developed to explain block of Na channels by local anesthetics. The preferential block of Na channels during depolarization helps explain the selectivity of DPH for neurons involved in seizure activity.

A revised view of cardiac sodium channel "blockade" in the long-QT syndrome.

Mutations in SCN5A, encoding the cardiac sodium (Na) channel, are linked to a form of the congenital long-QT syndrome (LQT3) that provokes lethal ventricular arrhythmias. These autosomal dominant mutations disrupt Na channel function, inhibiting channel inactivation, thereby causing a sustained ionic current that delays cardiac repolarization. Sodium channel-blocking antiarrhythmics, such as lidocaine, potently inhibit this pathologic Na current (I(Na)) and are being evaluated in patients with LQT3. The mechanism underlying this effect is unknown, although high-affinity "block" of the open Na channel pore has been proposed. Here we report that a recently identified LQT3 mutation (R1623Q) imparts unusual lidocaine sensitivity to the Na channel that is attributable to its altered functional behavior. Studies of lidocaine on individual R1623Q single-channel openings indicate that the open-time distribution is not changed, indicating the drug does not block the open pore as proposed previously. Rather, the mutant channels have a propensity to inactivate without ever opening ("closed-state inactivation"), and lidocaine augments this gating behavior. An allosteric gating model incorporating closed-state inactivation recapitulates the effects of lidocaine on pathologic I(Na). These findings explain the unusual drug sensitivity of R1623Q and provide a general and unanticipated mechanism for understanding how Na channel-blocking agents may suppress the pathologic, sustained Na current induced by LQT3 mutations.

Local anesthetics as effectors of allosteric gating. Lidocaine effects on inactivation-deficient rat skeletal muscle Na channels.

Time- and voltage-dependent local anesthetic effects on sodium (Na) currents are generally interpreted using modulated receptor models that require formation of drug-associated nonconducting states with high affinity for the inactivated channel. The availability of inactivation-deficient Na channels has enabled us to test this traditional view of the drug-channel interaction. Rat skeletal muscle Na channels were mutated in the III-IV linker to disable fast inactivation (F1304Q: FQ). Lidocaine accelerated the decay of whole-cell FQ currents in Xenopus oocytes, reestablishing the wild-type phenotype; peak inward current at -20 mV was blocked with an IC50 of 513 microM, while plateau current was blocked with an IC50 of only 74 microM (P < 0.005 vs. peak). In single-channel experiments, mean open time was unaltered and unitary current was only reduced at higher drug concentrations, suggesting that open-channel block does not explain the effect of lidocaine on FQ plateau current. We considered a simple model in which lidocaine reduced the free energy for inactivation, causing altered coupling between activation and inactivation. This model readily simulated macroscopic Na current kinetics over a range of lidocaine concentrations. Traditional modulated receptor models which did not modify coupling between gating processes could not reproduce the effects of lidocaine with rate constants constrained by single-channel data. Our results support a reinterpretation of local anesthetic action whereby lidocaine functions as an allosteric effector to enhance Na channel inactivation.

Ion channels as enzymes: analogy or homology?

Ion channels render nerve and muscle excitable. A typical channel protein can mediate the passive transfer of millions of ions per second across the membrane. Thus, channels catalyse the transmembrane flux of ions, fulfilling criteria traditionally associated with enzymes. Is this a semantic coincidence, or do channels and enzymes in fact rely upon similar structural principles? A general answer remains elusive given the paucity of crystallographic data on channels. Nevertheless, emerging evidence points to fundamental similarities between the pores of channels and the active sites of enzymes of resolved structure. Shared features include narrow clefts lined by protein loops, and specific binding of transition intermediates during catalysis. The often cited analogies between channels and enzymes might therefore reflect basic design homologies.

Role of the calcium-independent transient outward current I(to1) in shaping action potential morphology and duration.

The Kv4.3-encoded current (I:(Kv4.3)) has been identified as the major component of the voltage-dependent Ca(2+)-independent transient outward current (I:(to1)) in human and canine ventricular cells. Experimental evidence supports a correlation between I:(to1) density and prominence of the phase 1 notch; however, the role of I:(to1) in modulating action potential duration (APD) remains unclear. To help resolve this role, Markov state models of the human and canine Kv4.3- and Kv1.4-encoded currents at 35 degrees C are developed on the basis of experimental measurements. A model of canine I:(to1) is formulated as the combination of these Kv4.3 and Kv1.4 currents and is incorporated into an existing canine ventricular myocyte model. Simulations demonstrate strong coupling between L-type Ca(2+) current and I:(Kv4.3) and predict a bimodal relationship between I:(Kv4.3) density and APD whereby perturbations in I:(Kv4.3) density may produce either prolongation or shortening of APD, depending on baseline I:(to1) current level.

Probing the interaction between inactivation gating and Dd-sotalol block of HERG.

Potassium channels encoded by HERG underlie I:(Kr), a sensitive target for most class III antiarrhythmic drugs, including methanesulfonanilides such as Dd-sotalol. Recently it was shown that these drugs are trapped in the channel as it closes during hyperpolarization. At the same time, HERG channels rapidly open and inactivate when depolarized, and methanesulfonanilide block is known to develop in a use-dependent manner, suggesting a potential role for inactivation in drug binding. However, the role of HERG inactivation in class III drug action is uncertain: pore mutations that remove inactivation reduce block, yet many of these mutations also modify the channel permeation properties and could alter drug affinity through gating-independent mechanisms. In the present study, we identify a definitive role for inactivation gating in Dd-sotalol block of HERG, using interventions complementary to mutagenesis. These interventions (addition of extracellular Cd(2+), removal of extracellular Na(+)) modify the voltage dependence of inactivation but not activation. In normal extracellular solutions, block of HERG current by 300 micromol/L Dd-sotalol reached 80% after a 10-minute period of repetitive depolarization to +20 mV. Maneuvers that impeded steady-state inactivation also reduced Dd-sotalol block of HERG: 100 micromol/L Cd(2+) reduced steady-state block to 55% at +20 mV (P:<0.05); removing extracellular Na(+) reduced block to 44% (P:<0.05). An inactivation-disabling mutation (G628C-S631C) reduced Dd-sotalol block to only 11% (P:<0.05 versus wild type). However, increasing the rate of channel inactivation by depolarizing to +60 mV reduced Dd-sotalol block to 49% (P:<0.05 versus +20 mV), suggesting that the drug does not primarily bind to the inactivated state. Coexpression of MiRP1 with HERG had no effect on inactivation gating and did not modify Dd-sotalol block. We postulate that Dd-sotalol accesses its receptor in the open pore, and the drug-receptor interaction is then stabilized by inactivation. Whereas deactivation traps the bound methanesulfonanilide during hyperpolarization, we propose that HERG inactivation stabilizes the drug-receptor interaction during membrane depolarization.

Cardiac sodium channels (hH1) are intrinsically more sensitive to block by lidocaine than are skeletal muscle (mu 1) channels.

When lidocaine is given systemically, cardiac Na channels are blocked preferentially over those in skeletal muscle and nerve. This apparent increased affinity is commonly assumed to arise solely from the fact that cardiac Na channels spend a large fraction of their time in the inactivated state, which exhibits a high affinity for local anesthetics. The oocyte expression system was used to compare systematically the sensitivities of skeletal (mu 1-beta 1) and cardiac (hH1-beta 1) Na channels to block by lidocaine, under conditions in which the only difference was the choice of alpha subunit. To check for differences in tonic block, Na currents were elicited after 3 min of exposure to various lidocaine concentrations at -100 mV, a potential at which both hH1-beta 1 and mu 1-beta 1 channels were fully reprimed. Surprisingly, hH1-beta 1 Na channels were threefold more sensitive to rested-state block by lidocaine (402 +/- 36 microM, n = 4-22) than were mu 1-beta 1 Na channels (1,168 +/- 34 microM, n = 7-19). In contrast, the inactivated state binding affinities determined at partially depolarized holding potentials (h infinity approximately 0.2) were similar (Kd = 16 +/- 1 microM, n = 3-9 for hH1-beta 1 and 12 +/- 2 microM, n = 4-11 for mu 1-beta 1). Lidocaine produced more use-dependent block of peak hH1-beta 1 Na current elicited by trains of short-(10 ms) or long- (1 s) duration step depolarizations (0.5 Hz, -20 mV) than of mu 1-beta 1 Na current. During exposure to lidocaine, hH1-beta 1 channels recover from inactivation at -100 mV after a prolonged delay (20 ms), while mu 1-beta 1 channels begin repriming immediately. The overall time course of recovery from inactivation in the presence of lidocaine is much slower in hH1-beta 1 than in mu 1-beta 1 channels. These unexpected findings suggest that structural differences in the alpha subunits impart intrinsically different lidocaine sensitivities to the two isoforms. The differences in steady state affinities and in repriming kinetics are both in the correct direction to help explain the increased potency of cardiac Na channel block by local anesthetics.

Molecular architecture of the voltage-dependent Na channel: functional evidence for alpha helices in the pore.

The permeation pathway of the Na channel is formed by asymmetric loops (P segments) contributed by each of the four domains of the protein. In contrast to the analogous region of K channels, previously we (Yamagishi, T., M. Janecki, E. Marban, and G. Tomaselli. 1997. Biophys. J. 73:195-204) have shown that the P segments do not span the selectivity region, that is, they are accessible only from the extracellular surface. The portion of the P-segment NH(2)-terminal to the selectivity region is referred to as SS1. To explore further the topology and functional role of the SS1 region, 40 amino acids NH(2)-terminal to the selectivity ring (10 in each of the P segments) of the rat skeletal muscle Na channel were substituted by cysteine and expressed in tsA-201 cells. Selected mutants in each domain could be blocked with high affinity by externally applied Cd(2)+ and were resistant to tetrodotoxin as compared with the wild-type channel. None of the externally applied sulfhydryl-specific methanethiosulfonate reagents modified the current through any of the mutant channels. Both R395C and R750C altered ionic selectivity, producing significant increases in K(+) and NH(4)(+) currents. The pattern of side chain accessibility is consistent with a pore helix like that observed in the crystal structure of the bacterial K channel, KcsA. Structure prediction of the Na channel using the program PHDhtm suggests an alpha helix in the SS1 region of each domain channel. We conclude that each of the P segments undergoes a hairpin turn in the permeation pathway, such that amino acids on both sides of the putative selectivity filter line the outer mouth of the pore. Evolutionary conservation of the pore helix motif from bacterial K channels to mammalian Na channels identifies this structure as a critical feature in the architecture of ion selective pores.

A structural rearrangement in the sodium channel pore linked to slow inactivation and use dependence.

Voltage-gated sodium (Na(+)) channels are a fundamental target for modulating excitability in neuronal and muscle cells. When depolarized, Na(+) channels may gradually enter long-lived, slow-inactivated conformational states, causing a cumulative loss of function. Although the structural motifs that underlie transient, depolarization-induced Na(+) channel conformational states are increasingly recognized, the conformational changes responsible for more sustained forms of inactivation are unresolved. Recent studies have shown that slow inactivation components exhibiting a range of kinetic behavior (from tens of milliseconds to seconds) are modified by mutations in the outer pore P-segments. We examined the state-dependent accessibility of an engineered cysteine in the domain III, P-segment (F1236C; rat skeletal muscle) to methanethiosulfonate-ethylammonium (MTSEA) using whole-cell current recordings in HEK 293 cells. F1236C was reactive with MTSEA applied from outside, but not inside the cell, and modification was markedly increased by depolarization. Depolarized F1236C channels exhibited both intermediate (I(M); tau approximately 30 ms) and slower (I(S); tau approximately 2 s) kinetic components of slow inactivation. Trains of brief, 5-ms depolarizations, which did not induce slow inactivation, produced more rapid modification than did longer (100 ms or 6 s) pulse widths, suggesting both the I(M) and I(S) kinetic components inhibit depolarization-induced MTSEA accessibility of the cysteine side chain. Lidocaine inhibited the depolarization-dependent sulfhydryl modification induced by sustained (100 ms) depolarizations, but not by brief (5 ms) depolarizations. We conclude that competing forces influence the depolarization-dependent modification of the cysteine side chain: conformational changes associated with brief periods of depolarization enhance accessibility, whereas slow inactivation tends to inhibit the side chain accessibility. The findings suggest that slow Na(+) channel inactivation and use-dependent lidocaine action are linked to a structural rearrangement in the outer pore.

Charged residues between the selectivity filter and S6 segments contribute to the permeation phenotype of the sodium channel.

The deep regions of the Na(+) channel pore around the selectivity filter have been studied extensively; however, little is known about the adjacent linkers between the P loops and S6. The presence of conserved charged residues, including five in a row in domain III (D-III), hints that these linkers may play a role in permeation. To characterize the structural topology and function of these linkers, we neutralized the charged residues (from position 411 in D-I and its homologues in D-II, -III, and -IV to the putative start sites of S6) individually by cysteine substitution. Several cysteine mutants displayed enhanced sensitivities to Cd(2+) block relative to wild-type and/or were modifiable by external sulfhydryl-specific methanethiosulfonate reagents when expressed in TSA-201 cells, indicating that these amino acids reside in the permeation pathway. While neutralization of positive charges did not alter single-channel conductance, negative charge neutralizations generally reduced conductance, suggesting that such charges facilitate ion permeation. The electrical distances for Cd(2+) binding to these residues reveal a secondary "dip" into the membrane field of the linkers in domains II and IV. Our findings demonstrate significant functional roles and surprising structural features of these previously unexplored external charged residues.

Functional association of the beta 1 subunit with human cardiac (hH1) and rat skeletal muscle (mu 1) sodium channel alpha subunits expressed in Xenopus oocytes.

Native cardiac and skeletal muscle Na channels are complexes of alpha and beta 1 subunits. While structural correlates for activation, inactivation, and permeation have been identified in the alpha subunit and the expression of alpha alone produces functional channels, beta 1-deficient rat skeletal muscle (mu 1) and brain Na channels expressed in Xenopus oocytes do not gate normally. In contrast, the requirement of a beta 1 subunit for normal function of Na channels cloned from rat heart or human heart (hH1) has been disputed. Coinjection of rat brain beta 1 subunit cRNA with hH1 (or mu 1) alpha subunit cRNA into oocytes increased peak Na currents recorded 2 d after injection by 240% (225%) without altering the voltage dependence of activation. In mu 1 channels, steady state inactivation was shifted to more negative potentials (by 6 mV, p < 0.01), but the shift of 2 mV was not significant for hH1 channels. Nevertheless, coexpression with beta 1 subunit speeded the decay of macroscopic current of both isoforms. Ensemble average hH1 currents from cell-attached patches revealed that coexpression of beta 1 increases the rate of inactivation (quantified by time to 75% decay of current; p < 0.01 at -30, -40, and -50 mV). Use-dependent decay of hH1 Na current during repeated pulsing to -20 mV (1 s, 0.5 Hz) after a long rest was reduced to 16 +/- 2% of the first pulse current in oocytes coexpressing alpha and beta 1 subunits compared to 35 +/- 8% use-dependent decay for oocytes expressing the alpha subunit alone. Recovery from inactivation of mu 1 and hH1 Na currents after 1-s pulses to -20 mV is multiexponential with three time constants; coexpression of beta 1 subunit decreased all three recovery time constants. We conclude that the beta 1 subunit importantly influences the function of Na channels produced by coexpression with either the hH1 or mu 1 alpha subunits.

Functional consequences of lidocaine binding to slow-inactivated sodium channels.

Na channels open upon depolarization but then enter inactivated states from which they cannot readily reopen. After brief depolarizations, native channels enter a fast-inactivated state from which recovery at hyperpolarized potentials is rapid (< 20 ms). Prolonged depolarization induces a slow-inactivated state that requires much longer periods for recovery (> 1 s). The slow-inactivated state therefore assumes particular importance in pathological conditions, such as ischemia, in which tissues are depolarized for prolonged periods. While use-dependent block of Na channels by local anesthetics has been explained on the basis of delayed recovery of fast-inactivated Na channels, the potential contribution of slow-inactivated channels has been ignored. The principal (alpha) subunits from skeletal muscle or brain Na channels display anomalous gating behavior when expressed in Xenopus oocytes, with a high percentage entering slow-inactivated states after brief depolarizations. This enhanced slow inactivation is eliminated by coexpressing the alpha subunit with the subsidiary beta 1 subunit. We compared the lidocaine sensitivity of alpha subunits expressed in the presence and absence of the beta 1 subunit to determine the relative contributions of fast-inactivated and slow-inactivated channel block. Coexpression of beta 1 inhibited the use-dependent accumulation of lidocaine block during repetitive (1-Hz) depolarizations from -100 to -20 mV. Therefore, the time required for recovery from inactivated channel block was measured at -100 mV. Fast-inactivated (alpha + beta 1) channels were mostly unblocked within 1 s of repolarization; however, slow-inactivated (alpha alone) channels remained blocked for much longer repriming intervals (> 5 s). The affinity of the slow-inactivated state for lidocaine was estimated to be 15-25 microM, versus 24 microM for the fast-inactivated state. We conclude that slow-inactivated Na channels are blocked by lidocaine with an affinity comparable to that of fast-inactivated channels. A prominent functional consequence is potentiation of use-dependent block through a delay in repriming of lidocaine-bound slow-inactivated channels.

Repolarization abnormalities, arrhythmia and sudden death in canine tachycardia-induced cardiomyopathy.

OBJECTIVES: This study sought to determine whether the canine model of tachycardia-induced heart failure (HF) is an effective model for sudden cardiac death (SCD) in HF. BACKGROUND: Such a well established HF model that also exhibits arrhythmias and SCD, along with repolarization abnormalities that could trigger them, may facilitate the study of SCD in HF, which still eludes effective treatment. METHODS: Twenty-five dogs were VVI-paced at 250 beats/min for 3 to 5 weeks. Electrocardiograms were obtained, and left ventricular endocardial monophasic action potentials (MAPs) were recorded at six sites at baseline and after HF. Weekly Holter recordings were made with pacing suspended for 24 h. RESULTS: Six animals (24%) died suddenly, one with Holter-documented polymorphic ventricular tachycardia (VT). Holter recordings revealed an increased incidence of VT as HF progressed. Repolarization was significantly (p < 0.05) prolonged, as indexed by a corrected QT interval (mean [+/-SD] 311 +/- 25 to 338 +/- 25 ms) and MAP duration measured at 90% repolarization (MAPD90) (181 +/- 19 to 209 +/- 28 ms), and spatial MAPD90 dispersion rose by 40%. We further tested whether CsCl inhibition of repolarizing K+ currents, which are reportedly downregulated in HF, might preferentially prolong the MAPD90 in HF. With 1 mEq/kg body weight of CsCl, MAPD90 rose by 86 +/- 100 ms in dogs with HF versus only 28 +/- 16 ms in control animals (p = 0.002). Similar disparities in CsCl sensitivity were observed in myocytes isolated from normal and failing hearts. CONCLUSIONS: Tachycardia-induced HF exhibits malignant arrhythmia and SCD, along with prolonged, heterogeneous repolarization and heightened sensitivity to CsCl at chamber and cellular levels. Thus, it appears to be a useful model for studying mechanisms and therapy of SCD in HF.

Electrical and structural remodeling of the failing ventricle.

Heart failure (HF) is a complex disease that presents a major public health challenge to Western society. The prevalence of HF increases with age in the elderly population, and the societal disease burden will increase with prolongation of life expectancy. HF is initially characterized by an adaptive increase of neurohumoral activation to compensate for reduction of cardiac output. This leads to a combination of neurohumoral activation and mechanical stress in the failing heart that trigger a cascade of maladaptive electrical and structural events that impair both the systolic and diastolic function of the heart.

Cysteine mutagenesis in the voltage-dependent sodium channel structural insights and implications.

The superfamily of ion channel proteins comprise multisubunit transmembrane glycoproteins that are the fundamental electrical signaling molecules in the heart and other excitable tissues. The large size and hydrophobicity of these proteins present a formidable obstacle to the generation of a crystal structure. In lieu of a high-resolution structure, complementary methods have been used to study the structure function relationships of these essential excitability proteins. Molecular cloning and biophysical analysis of heterologously expressed wild-type and mutant channel proteins have provided insights into the structural basis of the essential channel functions of permeation and gating. This powerful combination of techniques also provides dynamic structural information regarding channel proteins not likely to be forthcoming from a crystal structure. (Trends Cardiovasc Med 1997;7:211-218). © 1997, Elsevier Science Inc.

Dispersion of ventricular activation and refractoriness in patients with idiopathic dilated cardiomyopathy.

We sought to evaluate the electrophysiologic substrate for ventricular arrhythmias and sudden cardiac death in patients with idiopathic dilated cardiomyopathy. When compared with controls, patients with cardiomyopathy had prolonged activation times, increased dispersion of activation and recovery, and prolonged duration of monophasic action potential recordings at 70%, but not at 90%, of repolarization.

A functional assay for paralytic shellfish toxins that uses recombinant sodium channels.

Saxitoxin (STX) and its derivatives are highly toxic natural compounds produced by dinoflagellates commonly present in marine phytoplankton. During algal blooms ("red tides"), shellfish accumulate saxitoxins leading to paralytic shellfish poisoning (PSP) in human consumers. PSP is a consequence of the high-affinity block of voltage-dependent Na channels in neuronal and muscle cells. PSP poses a significant public health threat and an enormous economic challenge to the shellfish industry worldwide. The standard screening method for marine toxins is the mouse mortality bioassay that is ethically problematic, costly and time-consuming. We report here an alternative, functional assay based on electrical recordings in cultured cells stably expressing a PSP target molecule, the STX-sensitive skeletal muscle Na channel. STX-equivalent concentration in the extracts was calibrated by comparison with purified STX, yielding a highly significant correlation (R=0.95; N=30) between electrophysiological determinations and the values obtained by conventional methods. This simple, economical, and reproducible assay obviates the need to sacrifice millions of animals in mandatory paralytic shellfish toxin screening programs.