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PubMed1986

Closed channel-open channel equilibrium of the sodium channel of nerve. Simple models of macromolecular equilibria.

The consistency of an electrodiffusion kinetics to describe the time-dependent opening of sodium channels of nerve suggests that motions over relatively long distances (on the atomic scale) are involved in the equilibrium as well. As a result, it is expected that a relatively large fraction of possible macromolecular conformations are unreactive. An equilibrium constant between locally reactive forms and the unreactive conformations is introduced. The consequences of this formalism is investigated in a square well potential, a harmonic potential, and a system consisting of two harmonic potentials with different spatial extents. The limits of knowledge from Nernstian behavior are shown. As an alternative to the Nernstian analysis, the experimental data of the sodium channel's quasi-equilibrium - the probability of the channel's being open as a function of voltage - can be described as resulting from motion caused by an electric field on a charge which is confined by a harmonic potential. A force constant is found from this analysis. (Such Hookian force constants cannot be found from spectroscopic experiments in condensed systems where the large-displacement vibrations are overdamped and, hence, spectroscopically unobservable). From the force constant, an approximate value of the Young's modulus can be calculated. The modulus' value falls in the range for rubber. As for rubbers, the restoring force is, then, expected to be mostly entropic rather than enthalpic in origin. Using the appropriate theory for linear chains of rubber and the Young's modulus, the approximate length of the chain causing the rubber-like force is calculated. The result is found to be near the length suggested for the hydrophilic chains that connect transmembrane sections of the sodium channel.

Animals↗
PubMed2000

The cytosolic termini of the beta- and gamma-ENaC subunits are involved in the functional interactions between cystic fibrosis transmembrane conductance regulator and epithelial sodium channel.

Epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) are co-localized in the apical membrane of many epithelia. These channels are essential for electrolyte and water secretion and/or reabsorption. In cystic fibrosis airway epithelia, a hyperactivated epithelial Na(+) conductance operates in parallel with defective Cl(-) secretion. Several groups have shown that CFTR down-regulates ENaC activity, but the mechanisms and the regulation of CFTR by ENaC are unknown. To test the hypothesis that ENaC and CFTR regulate each other, and to identify the region(s) of ENaC involved in the interaction between CFTR and ENaC, rENaC and its mutants were co-expressed with CFTR in Xenopus oocytes. Whole cell macroscopic sodium currents revealed that wild type (wt) alphabetagamma-rENaC-induced Na(+) current was inhibited by co-expression of CFTR, and further inhibited when CFTR was activated with a cAMP-raising mixture (CKT). Conversely, alphabetagamma-rENaC stimulated CFTR-mediated Cl(-) currents up to approximately 6-fold. Deletion mutations in the intracellular tails of the three rENaC subunits suggested that the carboxyl terminus of the beta subunit was required both for the down-regulation of ENaC by activated CFTR and the up-regulation of CFTR by ENaC. However, both the carboxyl terminus of the beta subunit and the amino terminus of the gamma subunit were essential for the down-regulation of rENaC by unstimulated CFTR. Interestingly, down-regulation of rENaC by activated CFTR was Cl(-)-dependent, while stimulation of CFTR by rENaC was not dependent on either cytoplasmic Na(+) or a depolarized membrane potential. In summary, there appear to be at least two different sites in ENaC involved in the intermolecular interaction between CFTR and ENaC.

Amiloride↗
PubMed2003

Asymmetric organization of the pore region of the epithelial sodium channel.

Epithelial sodium channels (ENaCs) are composed of three homologous subunits that have regions preceding the second transmembrane domain (also referred as pre-M2) that form part of the channel pore. To identify residues within this region of the beta-subunit that line the pore, we systematically mutated residues Gln(523)-Ile(536) to cysteine. Wild type and mutant mouse ENaCs were expressed in Xenopus oocytes, and a two-electrode voltage clamp was used to examine the properties of mutant channels. Cysteine substitutions of 9 of 13 residues significantly altered Li(+) to Na(+) current ratios, whereas only cysteine replacement of beta Gly(529) resulted in K(+)-permeable channels. Besides beta G525C, large increases in the inhibitory constant of amiloride were observed with mutations at beta Gly(529) and beta Ser(531) within the previously identified 3-residue tract that restricts K(+) permeation. Cysteine substitution preceding (beta Phe(524) and beta Gly(525)), within (beta Gly(530)) or following (beta Leu(533)) this 3-residue tract, resulted in enhanced current inhibition by external MTSEA. External MTSET partially blocked channels with cysteine substitutions at beta Gln(523), beta Phe(524), and beta Trp(527). MTSET did not inhibit alpha beta G525C gamma, although previous studies showed that channels with cysteine substitutions at the corresponding sites within the alpha- and gamma-subunits were blocked by MTSET. Our results, placed in context with previous observations, suggest that pore regions from the three ENaC subunits have an asymmetric organization.

Amiloride↗
PubMed2003

Co-localization of sodium channel Nav1.6 and the sodium-calcium exchanger at sites of axonal injury in the spinal cord in EAE.

Axonal degeneration contributes to the development of non-remitting neurological deficits and disability in multiple sclerosis, but the molecular mechanisms that underlie axonal loss in multiple sclerosis are not clearly understood. Studies of white matter axonal injury have demonstrated that voltage-gated sodium channels can provide a route for sodium influx into axons that triggers reverse operation of the Na(+)/Ca(2+) exchanger (NCX) and subsequent influx of damaging levels of intra-axonal calcium. The molecular identities of the involved sodium channels have, however, not been determined. We have previously demonstrated extensive regions of diffuse expression of Na(v)1.6 and Na(v)1.2 sodium channels along demyelinated axons in experimental allergic encephalomyelitis (EAE). Based on the hypothesis that the co-localization of Na(v)1.6 and NCX along extensive regions of demyelinated axons may predispose these axons to injury, we examined the expression of myelin basic protein, Na(v)1.2, Na(v)1.6, NCX and beta-amyloid precursor protein (beta-APP), a marker of axonal injury, in the spinal cord dorsal columns of mice with EAE. We demonstrate a significant increase in the number of demyelinated axons demonstrating diffuse Na(v)1.6 and Na(v)1.2 sodium channel immunoreactivity in EAE (92.2 +/- 2.1% of beta-APP positive axons were Na(v)1.6-positive). Only 38.0 +/- 2.9% of beta-APP positive axons were Na(v)1.2 positive, and 95% of these co-expressed Na(v)1.6 together with Na(v)1.2. Using triple-labelled fluorescent immunohistochemistry, we demonstrate that 73.5 +/- 4.3% of beta-APP positive axons co-express Na(v)1.6 and NCX, compared with 4.4 +/- 1.0% in beta-APP negative axons. Our results indicate that co-expression of Na(v)1.6 and NCX is associated with axonal injury in the spinal cord in EAE.

Amyloid beta-Protein Precursor↗
PubMed1996

Sodium channel slow inactivation and the distribution of sodium channels on skeletal muscle fibres enable the performance properties of different skeletal muscle fibre types.

Na+ currents (INa) and membrane capacitance were studied with the loose patch voltage clamp technique and action potential properties were studied with a two-electrode voltage clamp on the end-plate, at the end-plate border and on extrajunctional membrane of skeletal muscle fibres. Slow inactivation regulates the available INa and is operative at the resting potential of both rat and human fibres. At the resting potential, slow inactivation causes a greater reduction in INa in fast-than in slow-twitch fibres. The relative resistance of slow-twitch fibres to slow inactivation may enable slow-twitch fibres to remain tonically active. Na+ channel inactivation may provide a peripheral mechanism that limits the duration that fast-twitch fibres can fire at high rates to prevent injury associated with prolonged high-frequency contraction. Consequently, slow inactivation may enable fast-twitch fibres to operate phasically at high rates or slow-twitch fibres to fire continuously at lower rates. For both fast- and slow-twitch fibres. INa normalized to membrane area was greatest on the end-plate, intermediate on the end-plate border and smallest on extrajunctional membrane. When normalized to membrane capacitance. INa was the same on the end-plate and the end-plate border and smallest on extrajunctional membrane. For a given membrane region, INa was larger on fast- than on slow-twitch fibres. The higher density of Na+ channels near the end-plate increased the safety factor for neuromuscular transmission by lowering the action potential threshold and increasing the action potential rate of rise at the end-plate.

Action Potentials↗
PubMed1990

Bundles of amphipathic transmembrane alpha-helices as a structural motif for ion-conducting channel proteins: studies on sodium channels and acetylcholine receptors.

Channel proteins are transmembrane symmetric (or pseudosymmetric) oligomers organized around a central ionic pore. We present here a molecular model of the pore forming structures of two channel proteins with different primary structures and oligomeric size: the voltage-sensitive sodium channel and the nicotinic cholinergic receptor. We report low-energy arrangements of alpha-helical bundles calculated by semiempiricial potential energy functions and optimization routines and further refined using molecular dynamics. The ion-conducting pore is considered to be a symmetric or pseudosymmetric homooligomer of 3-5 amphipathic alpha-helices arranged such that the polar residues line a central hydrophilic pathway and the apolar residues face the hydrophobic bilayer interior. The channel lining exposes either charged (Asp, Glu, Arg, Lys) or polar-neutral (Ser, Thr) residues. A bundle of four parallel helices constrained to C4 symmetry, the helix axis aligned with the symmetry axis, and the helices constrained to idealized dihedral angles, produces a structure with a pore of the size inferred for the sodium channel protein (area approximately 16 A2). Similarly, a pentameric array optimized with constraints to maintain C5 symmetry and backbone torsions characteristic of alpha-helices adopts a structure that appears well suited to form the lining of the nicotinic cholinergic receptor (pore area approximately 46 A2). Thus, bundles of amphipathic alpha-helices satisfy the structural, energetic, and dynamic requirements to be the molecular structural motif underlying the function of ionic channels.

Amino Acid Sequence↗
PubMed2001

Differential modulation of sodium channel gating and persistent sodium currents by the beta1, beta2, and beta3 subunits.

Brain sodium channels are complexes of a pore-forming alpha subunit with auxiliary beta subunits, which are transmembrane proteins that modulate alpha subunit function. The newly cloned beta3 subunit is shown to be expressed broadly in neurons in the central and peripheral nervous systems, but not in glia and most nonneuronal cells. Beta1, beta2, and beta3 subunits are coexpressed in many neuronal cell types, but are differentially expressed in ventromedial nucleus of the thalamus, brain stem nuclei, cerebellar Purkinje cells, and dorsal root ganglion cells. Coexpression of beta1, beta2, and beta3 subunits with Na(v)1.2a alpha subunits in the tsA-201 subclone of HEK293 cells shifts sodium channel activation and inactivation to more positive membrane potentials. However, beta3 is unique in causing increased persistent sodium currents. Because persistent sodium currents are thought to amplify summation of synaptic inputs, expression of this subunit would increase the excitability of specific groups of neurons to all of their inputs.

Animals↗
PubMed2006

Reversal of neuropathic pain by alpha-hydroxyphenylamide: a novel sodium channel antagonist.

Sodium (Na) channel blockers are known to possess antihyperalgesic properties. We have designed and synthesized a novel Na channel antagonist, alpha-hydroxyphenylamide, and determined its ability to inhibit both TTX-sensitive (TTX-s) and TTX-resistant (TTX-r) Na currents from small dorsal root ganglion (DRG) neurons. alpha-Hydroxyphenylamide tonically inhibited both TTX-s and TTX-r Na currents yielding an IC(50) of 8.2+/-2.2 microM (n=7) and 28.9+/-1.8 microM (n=8), respectively. In comparison, phenytoin was less potent inhibiting TTX-s and TTX-r currents by 26.2+/-4.0% (n=8) and 25.5+/-2.0%, respectively, at 100 microM. alpha-Hydroxyphenylamide (10 microM) also shifted equilibrium gating parameters of TTX-s Na channels to greater hyperpolarized potentials, slowed recovery from inactivation, accelerated the development of inactivation and exhibited use-dependent block. In the chronic constriction injury (CCI) rat model of neuropathic pain, intraperitoneal administration of alpha-hydroxyphenylamide attenuated the hyperalgesia by 53% at 100mg/kg, without affecting motor coordination in the Rotorod test. By contrast, the reduction in pain behavior produced by phenytoin (73%; 100mg/kg) was associated with significant motor impairment. In summary, we report that alpha-hydroxyphenylamide, a sodium channel antagonist, exhibits antihyperalgesic properties in a rat model of neuropathic pain, with favorable sedative and ataxic side effects compared with phenytoin.

Action Potentials↗
PubMed2003

Brain sodium channels and central sodium-induced increases in brain ouabain-like compound and blood pressure.

OBJECTIVE: To assess the role of benzamil-sensitive sodium channels in the increases in brain ouabain-like compounds (OLC) and in blood pressure by cerebrospinal fluid (CSF) Na+. METHODS: Artificial CSF (aCSF) or Na+-rich (0.8 mol/l Na+) aCSF, either alone or combined with benzamil (at 1.2 and 4.0 microg/kg per h), were infused intracerebroventricularly (i.c.v.) at 5 microl/h to Wistar rats for 14 days and the effects on the brain and peripheral OLC and blood pressure were studied. OLC content was measured by enzyme-linked immunosorbent assay. RESULTS: In Wistar rats infused i.c.v. with aCSF, benzamil did not affect blood pressure or brain and peripheral OLC concentrations. I.c.v. infusion of Na+-rich aCSF increased systolic blood pressure (140 +/- 4 mmHg compared with 119 +/- 3 mmHg; P < 0.05). Benzamil fully blocked this increase. Na+-rich aCSF increased hypothalamic (23 +/- 3 ng/g tissue compared with 10 +/- 1 ng/g tissue; P < 0.05) and pituitary (233 +/- 35 ng/g tissue compared with 62 +/- 7 ng/g tissue; P < 0.05) contents of OLC. In contrast, Na+-rich aCSF decreased OLC in the adrenal gland (7 +/- 1 ng/g tissue compared with 21 +/- 3 ng/g tissue; P < 0.05) and plasma (0.5 +/- 0.04 ng/ml compared with 0.7 +/- 0.08 ng/ml; P < 0.05). Benzamil inhibited these responses of OLC to CSF sodium in a dose-related manner. CONCLUSIONS: These findings suggest that benzamil-sensitive brain sodium channels mediate the increase in brain OLC and the subsequent hypertension induced by increased CSF Na+.

Amiloride↗
PubMed1989

Immunochemical localization of amiloride-sensitive sodium channels in sodium-transporting epithelia.

The localization of amiloride-sensitive Na+ channels in Na+-transporting epithelia was examined using antibodies made against amiloride-binding Na+ channel protein purified from bovine kidney. The distribution of the channel protein was determined in thick frozen sections at the light-microscopic level using indirect immunofluorescence, and at the electron-microscopic level using immunogold labelling. In the cells of both the intact bovine collecting tubule and A6 confluent monolayers, only the luminal or apical-facing surface membranes showed staining. Sodium channel protein was characteristically localized on microvillar domains of the apical plasma membrane. Little or no basolateral membrane staining was evident. Channel protein was also absent from subapical vesicles and tight junctions, and was not found in bovine renal proximal tubules, cultured human secretory sweat coils, non-epithelial Chinese hamster ovary (CHO) cells or human skin fibroblasts. Trypsinization of intact A6 monolayers prior to cell fixation abolished specific staining with antibody. Pretreatment with amiloride protected against this loss of staining. Thus, our probes are specific for amiloride-binding Na+ channel protein, and this channel protein is largely or completely confined to the apical membrane of Na+-transporting epithelia. The level and distribution of specific immunostaining in A6 cells was unchanged by aldosterone treatment, although channel activity, as measured by short-circuit current, increased threefold. This result demonstrates that Na+ channel protein is ever present at the cell surface and exists in both an active and an inactive form. We find no evidence that stimulation of Na+ uptake by aldosterone involves recruitment of new channels from a cytoplasmic pool.

Aldosterone↗
PubMed1985

The calcium channel blocker nitrendipine blocks sodium channels in neonatal rat cardiac myocytes.

The dihydropyridine calcium channel blocker, nitrendipine, was studied for its effects on the sodium current of single cultured ventricular cells from neonatal rats. The patch-clamp method of recording whole cell currents was used, and sodium currents were isolated by suppressing potassium and calcium currents. Potassium currents were blocked by replacing potassium with cesium in the internal and external solutions and by adding tetraethylammonium chloride and 4-aminopyridine in the external solution; calcium current was blocked by replacing calcium with cobalt in the external solution. At low frequencies (0.1 Hz), nitrendipine reduced sodium currents without any significant change in the current-voltage relation. The block was dose dependent, and assuming a single occupancy model with complete block, had a half-maximum value of 3 X 10(-6) M at a holding potential of -80 mV where half the sodium channels are activatable. This value is within the range of the Kd's that have been reported for low-affinity dihydropyridine-binding sites found in cardiac sarcolemmal vesicles. In the presence of nitrendipine, the inactivation curve was shifted to hyperpolarized potentials. The block was greater with pulse intervals shorter than 1000 msec, and repriming was prolonged in the presence of the drug. These effects are similar to those of local anesthetics of the tertiary amine class, such as lidocaine. The block was relieved by the dihydropyridine agonist Bay K8644. The results are interpreted as indicating that dihydropyridines react with sodium channels.

Animals↗
PubMed1988

Formation of inositol phosphates in synaptoneurosomes of guinea pig brain: stimulatory effects of receptor agonists, sodium channel agents and sodium and calcium ionophores.

Carbamylcholine, norepinephrine, histamine and glutamate stimulated the formation of [3H]inositol phosphates in [3H]inositol-labelled synaptoneurosomes obtained from the cortex, striatum and hippocampus of the guinea pig. Synaptoneurosomes prepared from the cerebellum did not respond to receptor agonists. Agents that enhance the influx of sodium ions through voltage-sensitive channels, such as batrachotoxin, scorpion venom and pumiliotoxin B, or a sodium ionophore, monensin, stimulated the formation of [3H]inositol phosphates in synaptoneurosomes from all four regions of the brain. Neither calcium channel blockers nor receptor antagonists reduced the responses to batrachotoxin. Ionomycin, a calcium ionophore, also stimulated the formation of [3H]inositol phosphates in synaptoneurosomes from all four regions of the brain. A phorbol ester inhibited the formation of [3H]inositol phosphates, elicited by either receptor agonists or by sodium channel agents. The major [3H]inositol-labelled lipid in the synaptoneurosomes was phosphatidylinositol as analyzed by thin layer chromatography. While the hydrolysis of phosphatidylinositol elicited by carbamylcholine resulted in an increase of lipid-labelling with [3H]inositol, sodium channel agents caused a decrease in the incorporation of [3H]inositol. The results indicate that intracellular sodium may have a regulatory role in the turnover of phosphatidylinositol, and that, unlike the receptor-mediated responses, this regulation is present in all regions of the brain.

Adenosine Triphosphate↗
PubMed1985

Behaviour of chemically modified sodium channels in frog nerve supports a three-state model of inactivation.

Voltage clamp experiments were done on single myelinated nerve fibres of the frog, Rana esculenta, with 10 mM TEA+ in the external solutions to block potassium channels. Sodium current inactivation was measured in TEA-Ringer solution and after treatment with Anemonia sulcata toxin II (5 microM), internal iodate (20/40 mM), glutaraldehyde (10 mM), chloramine-T (0.6 mM), and 2,4,6-trinitrophenol (1 mM). The diphasic inactivation time course, observed in untreated membranes, is slowed by all these agents in a very similar way. Both time constants are increased and the proportion of inactivation components is changed favouring the slowly inactivating one. Trinitrophenol only slows inactivation, whereas in Anemonia toxin II, internal iodate, glutaraldehyde and chloramine-T inactivation becomes incomplete, so that a persistent current is flowing during depolarizations. None of these agents even at high concentrations however, totally removes inactivation. These modifications of inactivation time course are interpreted as changes of rate constants in a three-state inactivation model with one open and two closed states (o-c-c). After chemical treatment the access to the closed states is impeded and the transitions into the open state are accelerated. If the membrane is depolarized during drug application chloramine-T fails to modify inactivation. The curve relating the steady state inactivation parameter, h infinity, to the conditioning potential, V pp becomes non-monotonic in chloramine-T, i.e. dh infinity/dV pp greater than 0 for V pp greater than 60 mV. Trinitrophenol, which per se fails to produce a persistent current component, increases the persistent current in a fibre pretreated with chloramine-T.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Short-circuit current overshoot in epithelial sodium channels following apical sodium jump.

Following a jump in the sodium concentration of the solution bathing the apical surface of frog skin, the inward sodium current rises rapidly to a peak and then falls to a steady-state plateau. Lindemann suggested that this fall is due to rapid closing (in 2 to 3 s) of Na channels. However, the lack of a corresponding corner frequency in the sodium-noise spectrum indicates a much slower closing. We propose a compartmental mechanism for the overshoot: the inward Na current causes Na to accumulate in the intracellular region adjacent to the sodium channel--a virtual compartment--thereby decreasing the outside/inside [Na] ratio. As that ratio falls with rising [Na] in the virtual compartment, the force driving the current falls. The predictions of such a model have been curve-fitted to the time-course of the current overshoot. The differential equation describing the rate of change of [Na] in the virtual compartment has several time constants: a filling time for the compartment, a leakage time for escape of Na into the larger intracellular space, a mixing time in the apical bathing solution, and, of course, the channel-closing time. This curve fitting shows that channel closing becomes important only in the tail of the overshoot (> 15 s) with mean open times in a range from 7 s to 3 min. Similarly, the time-course of the current after washout of apical [Na] was fitted using the same differential equation, with the channel-closing time replaced with a channel-opening time. Other phenomena explainable by this compartmental model but not by fast channel closing include the open-circuit-potential overshoot, current overshoot through nystatin channels, and the less-than-59-mV-per-decade slopes of semilog plots of open-circuit potential vs. [Na].

Animals↗
PubMed2001

The intracellular segment of the sodium channel beta 1 subunit is required for its efficient association with the channel alpha subunit.

Sodium channels consist of a pore-forming alpha subunit and auxiliary beta 1 and beta 2 subunits. The subunit beta 1 alters the kinetics and voltage-dependence of sodium channels expressed in Xenopus oocytes or mammalian cells. Functional modulation in oocytes depends on specific regions in the N-terminal extracellular domain of beta 1, but does not require the intracellular C-terminal domain. Functional modulation is qualitatively different in mammalian cells, and thus could involve different molecular mechanisms. As a first step toward testing this hypothesis, we examined modulation of brain Na(V)1.2a sodium channel alpha subunits expressed in Chinese hamster lung cells by a mutant beta1 construct with 34 amino acids deleted from the C-terminus. This deletion mutation did not modulate sodium channel function in this cell system. Co-immunoprecipitation data suggest that this loss of functional modulation was caused by inefficient association of the mutant beta 1 with alpha, despite high levels of expression of the mutant protein. In Xenopus oocytes, injection of approximately 10,000 times more mutant beta 1 RNA was required to achieve the level of functional modulation observed with injection of full-length beta 1. Together, these findings suggest that the C-terminal cytoplasmic domain of beta 1 is an important determinant of beta1 binding to the sodium channel alpha subunit in both mammalian cells and Xenopus oocytes.

Animals↗
PubMed1999

Terbutaline increases open channel density of epithelial sodium channel (ENaC) in distal lung.

Neonatal and adult vertebrate respiration is facilitated by alveolar fluid and sodium (Na+) absorption driven by apical sodium channels (ENaC). ENaC are characterized in Xenopus laevis lung (XLL) epithelia using voltage clamping and fluctuation analysis to non-invasively examine macroscopic transepithelial current and resistance (I(SC), R(T)), single channel current (i(Na)) and total channel density (N(T)) responses to a beta adrenergic agonist (Terbutaline). Terbutaline addition to the basolateral bath of XLL increased Na entry to > 200% of control reflecting a doubling of open channel density (N(o). These data are consistent with the notion that XLL can serve as a useful model for investigation of distal lung ENaC response to agents of physiological interest.

Adrenergic beta-Agonists↗
PubMed2006

The voltage dependence of recovery from use-dependent block by QX-222 separates mechanisms for drug egress in the cardiac sodium channel.

In neuronal sodium channels of squid giant axons, recovery from QX-222 block is slowed by hyperpolarization. However, in ventricular cells, hyperpolarization speeds recovery. Previously, we showed that isoform-specific residues in the external side of the cardiac sodium channel isoform (D1P-loop C373 and D4S6 T1752) influence use-dependent block (UDB) by lidocaine. To determine whether these isoformspecific residues contribute to the contrasting voltage-dependent recovery observed in ventricular myocytes, we measured recovery rates from UDB by QX-222 at holding potentials of 120, 140, 160 and 180 mV for wild-type cardiac channel (WT), the mutants C373Y (CY) and T1752V (TV), and C373Y/T1752V (CY/TV). Unlike neuronal channels, cardiac sodium channels recovered from QX block faster at hyperpolarized potentials. All mutations slowed QX-222 recovery, with the greatest rate reduction observed for the double mutant, indicating that the isoform-specific residues define external drug paths. The recovery rates varied linearly with voltage over the range tested, and we used the slopes of rate versus voltage plots to quantify voltage dependence. The TV mutation caused reduction in recovery rates without changing the slope, indicating that the mutation closed a voltage-independent egress path. The CY mutation, however, flattened the slope and reduced the voltage dependence of recovery. In addition, the reduction in rate caused by CY/TV is less than the sum of those for CY and TV, suggesting that the impacts of these two residues are interrelated. Therefore, we propose that the isoform-specific residues C373 and T1752 change recovery from UDB by distinct mechanisms but determine a common drug egress path.

Cell Line↗