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The sodium channel β1 subunit is non-covalently associated with the pore-forming α-subunits, and has been proposed to act as a modulator of channel activity, regulator of channel cell surface expression and cell adhesion molecule. Its importance is evident since mutations of the β1 subunit cause neurologic and cardiovascular disorders. The first described β1 subunit mutation is the C121W, that is related to generalized epilepsy with febrile seizures plus (GEFS+), a childhood genetic epilepsy syndrome. This mutation changed a conserved cysteine residue in position 121 into a tryptophan, putatively disrupting a disulfide bridge that should normally maintain the β1 extracellular immunoglobulin-like fold. Using the 2-D-diagonal-SDS-PAGE technique, we demonstrated the existence of this putative disulfide bridge in the Ig-like extracellular domain of the β1 subunit and its disruption in the epileptogenic C121W mutant., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Voltage-gated sodium channels are composed of one α subunit and one or more auxiliary β subunits. A standard reverse transcription-polymerase chain reaction assay was used to detect the mRNAs encoding for seven α subunits (Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.9) and for the two non-covalently linked β1 and β3 auxiliary subunits in five different cell lines from rat, mouse and human origin. A semi-quantitavive RT-PCR analysis allowed to evaluate in each cell line, the relative expression level of each NaCh subunit previously detected. The expression profile of the cell lines was compared with that obtained from rat and mouse neural, skeletal muscle and cardiac tissues. This data provide a standard for the study of the modulation of the sodium channel expression in mammalian excitable tissues.

Voltage-gated sodium channels, comprised of a pore-forming alpha-subunit and additional regulatory (beta) subunits, play a critical role in regulation of neuronal excitability. Mechanisms of regulation of beta-subunits remain elusive. We have tested the functional effects of beta1 sodium channel subunit on surface charges as a mechanism for channel modulation. HEK-293 cell lines permanently transfected with the sole rat skeletal muscle sodium channel alpha-subunit (Nav1.4), or co-expressing the sodium channel alpha-subunit and beta1-subunit were studied with the whole-cell mode of the patch-clamp technique. At physiological extracellular Ca2+ concentration (2 mM), expression of beta1-subunit did not produce any significant effect on the voltage-dependent properties of sodium currents. However, a shift of half-activation potentials of sodium channel by changing the extracellular Ca2+ was potentiated when beta1 was co-expressed with alpha-subunit. In contrast, the expression of beta1-subunit did not affect the Ca2+ binding to the open or to the closed sodium channel pore, difference of the effect provoked by extracellular Ca2+ could therefore be attributed to an increased in negative surface charge determined by the presence of beta1-subunit. These data are in agreement with the hypothesis of a modulation of the sodium current by the expression of the highly sialylated beta1-subunit, which would alter the channel gating by increasing the density of surface negative charges in the vicinity of the sodium channel voltage sensing machinery.

We studied the properties of a sodium channel comprised only of S5-P-S6 region of the rat sodium channel alpha-subunit Nav1.4 (micro1pore). Results obtained in HEK cell lines permanently transfected with the sodium channel alpha-subunit or with the micro1pore were compared with data of the native HEK cells. Sodium channel blockers, tetrodotoxin and tetracaine, protect cells transfected with the complete sodium channel against death produced by incubation with veratridine. Veratridine-induced cell death in cell lines expressing the micro1pore construct is antagonised by tetracaine, but not by tetrodotoxin. Whole-cell conductance also increases in the presence of veratridine in micro1pore transfected cells and tetracaine inhibits these currents. Our pharmacological and electrophysiological data suggest that micro1pore keeps binding sites for veratridine and tetracaine, but not for TTX, and reconstitutes the permeation pathway for Na+ ions.

The blockage of skeletal muscle sodium channels by tetrodotoxin (TTX) and saxitoxin (STX) have been studied in CHO cells permanently expressing rat Nav1.4 channels. Tonic and use-dependent blockage were analyzed in the framework of the ion-trapped model. The tonic affinity (26.6 nM) and the maximum affinity (7.7 nM) of TTX, as well as the "on" and "off" rate constants measured in this preparation, are in remarkably good agreement with those measured for Nav1.2 expressed in frog oocytes, indicating that the structure of the toxin receptor of Nav1.4 and Nav1.2 channels are very similar and that the expression method does not have any influence on the pore properties of the sodium channel. The higher affinity of STX for the sodium channels (tonic and maximum affinity of 1.8 nM and 0.74 nM respectively) is explained as an increase on the "on" rate constant (approximately 0.03 s(-1) nM(-1)), compared to that of TTX (approximately 0.003 s(-1) nM(-1)), while the "off" rate constant is the same for both toxins (approximately 0.02 s(-1)). Estimations of the free-energy differences of the toxin-channel interaction indicate that STX is bound in a more external position than TTX. Similarly, the comparison of the toxins free energy of binding to a ion-free, Na(+)- and Ca(2+)-occupied channel, is consistent with a binding site in the selectivity filter for Ca(2+) more external than for Na(+). This data may be useful in further attempts at sodium-channel pore modeling.

Sodium currents in cell lines transfected with the sole alpha-subunit, or constitutively expressing sodium channels, have an inactivation that is always prevalently mono-exponential. Differently, expression of alpha-subunit in Xenopus oocytes exerts slow inactivating currents with biphasic decay, while simultaneous co-transfection of alpha and beta1 restores a mono-exponential (normal) inactivation. A hypothesis for such differences is that an endogenous presence of beta1 or beta1-alternative splicing, beta1A, in cells could account for the normal inactivation. To test this hypothesis and to evaluate the role for the beta1A, we inhibited the expression of beta1/beta1A by antisense oligonucleotides on Nav1.4-transfected human embryonic cell line 293 (HEK) cells. Reduction of beta1/beta1A produces no significant functional effects in Nav1.4-HEK. This result invalidates the hypothesis that the lack of slow-mode in cell lines is simply due to a constitutive expression of beta1/beta1A.

The syndrome of generalized epilepsy with febrile seizure plus (GEFS+) is associated with a single point mutation on the gene SCN1B that results in a substitution of the cysteine 121 with a tryptophane in the sodium channel beta 1-subunit protein. We have studied, in the HEK cells permanently transfected with the skeletal muscle sodium channel alpha-subunit (SkM1), the effects of a transient transfection of the wild type (WT) or C121W mutant beta 1-subunit. Coexpression of the WT beta 1 produces two effects on the sodium currents expressed in mammalian cells: the increase in the density of sodium channels, and the modulation of the inactivation of the sodium currents, inducing a hastening of the recovery from the inactivation. This modulation is less severe as observed when sodium channels are expressed in frog oocytes. We have observed that mutant C121W lacks this modulatory property, but maintains its property to increase the current density. Our observation suggests a possible involvement of this lack of modulation in the development of the GEFS+, providing the first hypothesis based on the observation of the functional properties of the beta 1-subunit C121W mutant in mammalian cells, which certainly represents a more physiological preparation, instead of in Xenopus oocytes, where the modulatory properties of the beta 1-subunit are artificially amplified.

The syndrome of generalized epilepsy with febrile seizures plus type 1 (GEFS+) has been associated to the gene SCN1B coding for the sodium channel beta1 subunit (Wallace, R. H. et al. (1998) Nature Genetics 19, 366-370). In patients, a mutation of the cysteine 121 to trpyptophane (C121W) would cause a lack of modulatory activity of the beta1 subunit on sodium channels expressed in the brain, rendering neurons hyperexcitable. We have confirmed that the normal beta1-modulation of type-IIA adult brain alpha subunits (BIIA) expressed in frog oocytes is defective in C121W. We observed that the mixture of wild-type and mutant beta1 subunits is less effective than wild-type alone, suggesting that the mutant beta1 subunit does bind the alpha subunit. However, we also observed a similar lack of modulation by C121W of the in adult skeletal muscle alpha subunit (SkM1). This finding is in contrast with the simple idea that the mutational effect observed in the oocyte expression system is the principal physiopathological correlate of GEFS+, because no skeletal muscle symptoms have been reported in GEFS+ patients. We conclude that the manifestation of the pathological phenotype is conditioned by the presence of susceptibility genes and/or that the frog oocyte expression system is inadequate for the study of the mutant beta1 subunit physiopathology., (Copyright 2001 Academic Press.)

Several observations suggest an interaction of the sodium channel alpha-subunit with the cytoskeletal structures. However, there is a wide variability in the results of experiments of heterologous expression in Xenopus oocytes and studies on mammalian cells are sometimes contradictory. In general, there has been no direct demonstration that ad hoc large perturbations of the cytoskeleton modify the intrinsic properties of the sodium channels expressed endogenously or heterologously in plasma membranes. We have studied in CHO cells transfected with the rat muscle sodium channel alpha-subunit the effects of two substances expected to produce drastic perturbations of the cytoskeletal structure: Cytochalasin-D, which depolymerizes microfilaments, and Colchicine, which inhibits the microtubules polymerization. We observed no significant differences in the voltage dependence, kinetic parameters and surface density of the expressed sodium channels after treatment of the cells with these substances. We conclude that the two known main components of the cytoskeleton do not interfere directly with the sodium channel function or with the heterologous expression of channels in the cell membrane., (Copyright 2000 Academic Press.)

The expression of the sole alpha-subunit of muscle or brain sodium channels in frog oocytes mediates currents with a bimodal inactivation with an abnormal slow mode that is strongly depressed only by co-expression of the beta1-subunit. In contrast, in the expression of the alpha-subunit in the human embryonic kidney cell line, HEK-293, the slow mode is almost absent, suggesting an endogenous expression of the beta1-subunit. We have tested this hypothesis by reverse transcriptase-polymerase chain reaction. We found an abundant expression of mRNA encoding the beta1A splicing of the putative regulatory sodium channel subunit but no mRNA encoding the beta1-subunit in HEK cells. This finding is consistent with the idea that the endogenous beta1A-subunit is sufficient for suppressing the slowly inactivating mode of sodium currents by co-assembly with alpha-subunits, and calls attention to the reliability of effects attributed in HEK cells to alpha-beta1 co-expression.

Three groups of mutations of the alpha subunit of the rat skeletal muscle sodium channel (rSkM1), homologous to mutations linked to human muscle hereditary diseases, have been studied by heterologous expression in frog oocytes: S798F, G1299E, G1299V, and G1299A, linked with potassium-aggravated myotonia (PAM); T1306M, R1441C and R1441P, linked with paramyotonia congenita (PC); T698M and M1353V, linked with the hyperkalemic periodic paralysis (HyPP). Wild-type rSkM1 channels (WT) show two gating modes, M1 and M2, which differ mainly in the process of inactivation. The naturally most representative mode M1 is tenfold faster and develops at approximately 30 mV less depolarized potentials. A common feature of myopathy-linked mutants is an increase in the mode M2 probability, P(M2), but phenotype-specific alterations of voltage-dependence and kinetics of inactivation of both modes are also observed. The coexpression of the sodium channel beta1 subunit, which has been studied for WT and for the five best expressing mutants, generally caused a threefold reduction of P(M2) without changing the properties of the individual modes. This indicates that the mutations do not affect the alpha - beta1 interaction and that the phenotypic changes in P(M2) observed for the enhanced mode M2 behavior of the sole alpha subunits, although largely depressed in the native tissue, are likely to be the most important functional modification that causes the muscle hyperexcitability observed in all patients carrying the myotonic mutations. The interpretation of the more phenotype-specific changes revealed by our study is not obvious, but it may offer clues for understanding the different clinical manifestations of the diseases associated with the various mutations.

We have studied the expression in frog oocytes of the alpha subunit of the rat skeletal muscle sodium channel mutation T1306M, homologous to the mutation T1313M of the human isoform that causes the muscular hereditary disease paramyotonia congenita. Wild-type (WT) channels show a bimodal behavior, with two gating modes characterized by inactivation time constants that differ at least by one order of magnitude and with voltage dependencies shifted by +27 mV in the slow mode (M2) relative to the fast (M1) mode. In the myopathy-linked mutant the propensity of the channel for the mode M2 is increased fourfold and the kinetics and voltage dependence of inactivation in both modes are altered. In mode M1, the onset of inactivation is faster and the recovery from inactivation is slower whereas both processes are slowed in mode M2. The half-inactivation potential of both modes is shifted by the mutation to positive potentials. Coexpression of beta subunit causes a threefold reduction of the M2 propensity of both WT and T1306M channels, with small changes in the voltage dependency and kinetic properties of inactivation. All the changes are consistent with the hyperexcitability of the muscle fibers observed in patients affected by potassium-aggrevated myotonia (PAM).

We have studied the expression in frog oocytes of the alpha-subunit of the rat skeletal muscle sodium channel mutation S798F, homologous to the mutation S804F of the human isoform, that causes potassium aggravated myotony (PAM), a muscular hereditary disease in humans. Wild type channels show a bimodal inactivation, with two gating modes that inactivate with time constants that differ at least by one order of magnitude and a steady steady-state voltage dependence of the slow mode shifted by +27 mV relative to that of the fast mode. In the myopathy-linked mutant the propensity of the channel to gate in the slow mode is significantly increased and there are alterations in the inactivation properties of both modes. The half inactivation potential of the fast mode is shifted negatively, and the inactivation kinetics of both modes are slower, with an apparent shift in their voltage dependence. The changes on the inactivation properties of the mutant channel may be related with the muscle fibre hyperexcitability observed patients affected by PAM.

Calcium ions block the open configuration and antagonise the tonic binding of TTX to the closed state of sodium channels in very different ranges of extracellular concentration, [Ca]o. We measured the open-state block in channels expressed in Xenopus oocytes by alpha-subunits from rat brain (rBIIa) or adult rat skeletal muscle (rSkM1). Recordings of instantaneous tail-currents from cell-attached macro patches show that the binding of Ca2+ to the blocking site has a dissociation constant of about 20 mM at 0 mV and senses about 30% of the membrane potential drop, whereas the concentration of half-inhibition of TTX-binding is less than 1 mM and voltage-insensitive. Assuming that both effects involve a single binding site, a simple model predicts that the state-dependency of the dissociation constant entails positive shifts of activation and faster kinetics of deactivation at increasing [Ca]o. The shifts of activation measured for rBIIA and rSkM1 channels are comparable in size to those predicted by the model, which accounts also for the observed larger shifts of the rBIIA-mutant K226Q as a consequence of its reduced voltage-sensitivity. Shifts attributable to surface-charge screening effects seem smaller in the oocyte than in native cell-membranes. The experimental [Ca]o-dependence of deactivation kinetics is also consistent with the model and with the idea that Ca(2+)-binding changes to the same extent, but in opposite directions, the activation free-energies of both opening and closing transitions.

We have studied S4-proline mutants of the rat brain sodium channel II. In mutant A224P one proline was added on the S4 segment of repeat I, and in mutant P1313V a proline was removed from the segment S4 of the repeat II. In both mutants, the activation curve was shifted to more positive potentials, without changing the steepness of the voltage dependence. The time course of inactivation, consisting of two exponential components, was similar in the wild type and in mutant A224P. Differently, the decay of the current in mutant P1313V had only one component, with a time constant similar to that of the fast component of wild type channels. This change in kinetics was accompanied in mutant P1313V by a change in the voltage dependence of the apparent steady-state inactivation. We conclude that the addition or deletion of prolines in segment S4 does not affect significantly the activation of sodium channels, but alters their mode of inactivation.

The aim of this study was to determine from macroscopic current analysis how intracellular magnesium ions, Mgi2+, interfere with sodium channels of mammalian neurones. It is reported here that permeation across the sodium channel is voltage- and concentration-dependently reduced by Mgi2+. This results in a general reduction of sodium membrane conductance and an outward sodium peak current at large positive potentials. 30 mM Mgi2+ leads ot a negative shift of voltage dependence of sodium channel gating parameters, probably due to the surface potential change of the membrane. This shift alone is, however, insufficient to explain the reduction of outward sodium currents. The blockage by Mgi2+ is decreased upon increasing intracellular or extracellular Na+ concentration, which suggests that Mgi2+ interferes with sodium permeation by competitively occupying sodium channels. Using a kinetic model to describe the sodium permeation, the dissociation constant (at zero membrane potential) of Mgi2+ for the sodium channel has been calculated to be 8.65 +/- 1.51 mM, with its binding site located at 0.26 +/- 0.05 electrical distance from the inner membrane. This dissociation constant is smaller than that of Nai+, which is 83.76 +/- 7.60 mM with its binding site located at 0.75 +/- 0.23. The low dissociation constant of Mgi2+ reflects its high affinity for the sodium channel.

Sodium currents were studied in granule cells dissociated from rat cerebellum. Macroscopic currents were recorded using the patch-clamp technique. Sodium currents, which are TTX sensitive, reached a maximum peak value of 0.42 +/- 0.08 pA/microns2 at 18.4 +/- 2.2 mV (n = 6). Activation and inactivation kinetics and steady-state properties were described in terms of Hodgkin and Huxley parameters. The properties of sodium channels in cultured rat cerebellar granule cells are very similar to those reported for various neural preparations.

Ionic and gating currents from voltage-gated sodium channels were recorded in mouse neuroblastoma cells using the path-clamp technique. Displacement currents were measured from whole-cell recordings. The gating charge displaced during step depolarizations increased with the applied membrane potential and reached saturating levels above 20 mV. Prolonged large depolarizations produced partial immobilization of the gating charge, and only about one third of the displaced charge was quickly reversed upon return to negative holding potentials. The activation and inactivation properties of macroscopic sodium currents were characterized by voltage-clamp analysis of large outside-out patches and the single-channel conductance was estimated from non-stationary noise analysis. The general properties of the sodium channels in mouse neuroblastoma cells are very similar to those previously reported for various preparations of invertebrate and vertebrate nerve cells.

Voltage-dependent sodium channels are membrane proteins essential for cell excitability. They are composed by a pore-forming α-subunit, encoded in mammals by up to nine different genes, and four different ancillary β-subunits. The expression pattern of the α subunit isoforms confers the distinctive functional and pharmacological properties to different excitable tissues. β-Subunits are important modulators of channel function and expression. Mutation C121W of the β1-subunit causes an autosomal dominant epileptic syndrome without cardiac symptoms. In neuroectoderm GH3 and cardiac H9C2 cells, the over-expression of β1 subunit augments α subunit mRNA and protein levels as well as sodium current density. Interestingly, the introduction of the epileptogenic C121W-β1 subunit produces additional changes in the α-subunit expression pattern of H9C2 cells, leaving unaltered the sodium channel isoform composition of GH3 cells. The challenge of the present work was to identify those genes that were differentially expressed in response to WT- or C121W-β1 subunit over-expression in the two rat cell lines under analysis. Hence, we analyzed the total mRNA extracted from control-untransfected and from WT- and C121W-β1-transfected GH3 and H9C2 cells by DNA-microarray. We found that, in agreement with their different embryonal origin, the over-expression of WT- and C121W-β1 subunits modifies the expression of different gene sets in GH3 and H9C2 cells. Focusing on the effects of the C121W mutation, we found that it causes the modification of 214 genes, most of them were down-regulated (202) in GH3 cells; on the contrary, it determined the up-regulation of only five genes in H9C2 cells. Interestingly, most genes modified by the C121W β1 subunit are involved in pivotal processes of the cell such as cellular communication and protein expression. Our results confirm the important role of the sodium channel β1 subunit in the control of NaCh gene expression, and highlight once more the tissue-specific effect of the C121W mutation. [ABSTRACT FROM AUTHOR]

Abstract Calcium ions block the open configuration and antagonise the tonic binding of TTX to the closed state of sodium channels in very different ranges of extracellular concentration, [Ca][sub o]. We measured the open-state block in channels expressed in Xenopus oocytes by alpha-subunits from rat brain (rBIIa) or adult rat skeletal muscle (rSkM1). Recordings of instantaneous tail-currents from cell-attached macro patches show that the binding of Ca[sup 2+] to the blocking site has a dissociation constant of about 20 mM at 0 mV and senses about 30% of the membrane potential drop, whereas the concentration of half-inhibition of TTX-binding is less than 1 mM and voltage-insensitive. Assuming that both effects involve a single binding site, a simple model predicts that the state-dependency of the dissociation constant entails positive shifts of activation and faster kinetics of deactivation at increasing [Ca]o. The shifts of activation measured for rBIIA and rSkMI channels are comparable in size to those predicted by the model, which accounts also for the observed larger shills of the rBIIA-mutant K226Q as a consequence of its reduced voltage-sensitivity. Shifts attributable to surface-charge screening effects seem smaller in the oocyte than in native cell-membranes. The experimental [Ca]o-dependence of deactivation kinetics is also consistent with the model and with the idea that Ca[sup 2+]-binding changes to the same extent, but in opposite directions, the activation free-energies of both opening and closing transitions. [ABSTRACT FROM AUTHOR]