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Mechanosensitivity of NaV1.5 sodium channels is regulated by specific -subunits In their paper entitled " 1 and 3 subunits amplify mechanosensitivity of the cardiac voltage-gated sodium channel Na SB V sb 1.5", the authors describe whole-cell patch-clamp recordings on human embryonic kidney (HEK) cells expressing Na SB V sb 1.5 -subunits either alone or in combination with 1 or 3, and expose the cells to shear stress generated by a standardized perfusion system. Using this approach, they confirmed that mechanical stress accelerates activation and inactivation of Na SB V sb 1.5 and demonstrate that only the presence of 3 but not 1 enhances the mechano-induced acceleration of channel activation and inactivation. [Extracted from the article]

Established risk factors for the metabolic syndrome as diabetes and arterial hypertension are believed to be the cause of arteriosclerosis and subsequently following diseases like coronary heart disease, apoplexy, or chronic renal failure. Based on broad evidence from the already available experimental literature and clinical experience, an alternative hypothesis is presented that puts an increased vessel and organ stiffness to the beginning of the pathophysiological scenario. The stiffness itself is caused by a persistent activation of mechano-sensitive cation channels like the epithelial/endothelial sodium channel. A further enhancement takes place by proteins like JACD and RhoA coupled phospholipase C coupled G-protein receptors and integrins. A self-enhancing positive feedback loop by activation of YAP/TAZ signaling is a further central pillar of this theory. Further investigations are necessary to verify this hypothesis. If this hypothesis could be confirmed fundamental changes regarding the pharmacologic therapy of the diseases that are currently summarizes as metabolic syndrome would be the consequence. [ABSTRACT FROM AUTHOR]

How phosphorylation of the epithelial sodium channel (ENaC) contributes to its regulation is incompletely understood. Previously, we demonstrated that in outside-out patches ENaC activation by serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) was abolished by mutating a serine residue in a putative SGK1 consensus motif RXRXX(S/T) in the channel's α-subunit (S621 in rat). Interestingly, this serine residue is followed by a highly conserved proline residue rather than by a hydrophobic amino acid thought to be required for a functional SGK1 consensus motif according to invitro data. This suggests that this serine residue is a potential phosphorylation site for the dual-specificity tyrosine phosphorylated and regulated kinase 2 (DYRK2), a prototypical proline-directed kinase. Its phosphorylation may prime a highly conserved preceding serine residue (S617 in rat) to be phosphorylated by glycogen synthase kinase 3 β (GSK3β). Therefore, we investigated the effect of DYRK2 on ENaC activity in outside-out patches of Xenopus laevis oocytes heterologously expressing rat ENaC. DYRK2 included in the pipette solution significantly increased ENaC activity. In contrast, GSK3β had an inhibitory effect. Replacing S621 in αENaC with alanine (S621A) abolished the effects of both kinases. A S617A mutation reduced the inhibitory effect of GKS3β but did not prevent ENaC activation by DYRK2. Our findings suggest that phosphorylation of S621 activates ENaC and primes S617 for subsequent phosphorylation by GSK3β resulting in channel inhibition. In proof-of-concept experiments, we demonstrated that DYRK2 can also stimulate ENaC currents in microdissected mouse distal nephron, whereas GSK3β inhibits the currents. [ABSTRACT FROM AUTHOR]

The binding ofH-benzamil to homogenates of epithelium removed from the colon and coprodaeum of hens was studied. A low capacity high affinity binding component was detected. The binding constants for benzamil and amiloride to this component were similar to those obtained when these ligands were used to inhibit transport in intact epithelia. The mean potency ratio of benzamil to amiloride was 12.8 measured by binding compared with 11.6 from functional inhibition. Binding activity was present in tissues taken from animals fed on low sodium diets and those containing a normal sodium content. In the presence of sodium the affinity of benzamil was slightly reduced, but only in tissues taken from animals on a low salt diet. Only a small fraction of the total binding activity was present in the apical surface of low salt tissues indicating that in homogenates a small percentage of the total activity is associated with functional sodium entry sites. It is suggested that the major part of the binding activity detected in homogenates represents components of the sodium ion translocation mechanism which are en route for the apical membrane. [ABSTRACT FROM AUTHOR]

Chronic pain is a global problem affecting up to 20% of the world's population and has a significant economic, social and personal cost to society. Sensory neurons of the dorsal root ganglia (DRG) detect noxious stimuli and transmit this sensory information to regions of the central nervous system (CNS) where activity is perceived as pain. DRG neurons express multiple voltage-gated sodium channels that underlie their excitability. Research over the last 20 years has provided valuable insights into the critical roles that two channels, NaV1.7 and NaV1.9, play in pain signalling in man. Gain of function mutations in NaV1.7 cause painful conditions while loss of function mutations cause complete insensitivity to pain. Only gain of function mutations have been reported for NaV1.9. However, while most NaV1.9 mutations lead to painful conditions, a few are reported to cause insensitivity to pain. The critical roles these channels play in pain along with their low expression in the CNS and heart muscle suggest they are valid targets for novel analgesic drugs. [ABSTRACT FROM AUTHOR]

Cav1.3 L-type Ca2+ channels (LTCCs) in cochlear inner hair cells (IHCs) are essential for hearing as they convert sound-induced graded receptor potentials into tonic postsynaptic glutamate release. To enable fast and indefatigable presynaptic Ca2+ signaling, IHC Cav1.3 channels exhibit a negative activation voltage range and uniquely slow inactivation kinetics. Interaction with CaM-like Ca2+-binding proteins inhibits Ca2+-dependent inactivation, while the mechanisms underlying slow voltage-dependent inactivation (VDI) are not completely understood. Here we studied if the complex formation of Cav1.3 LTCCs with the presynaptic active zone proteins RIM2α and RIM-binding protein 2 (RBP2) can stabilize slow VDI. We detected both RIM2α and RBP isoforms in adult mouse IHCs, where they co-localized with Cav1.3 and synaptic ribbons. Using whole-cell patch-clamp recordings (tsA-201 cells), we assessed their effect on the VDI of the C-terminal full-length Cav1.3 (Cav1.3L) and a short splice variant (Cav1.342A) that lacks the C-terminal RBP2 interaction site. When co-expressed with the auxiliary β3 subunit, RIM2α alone (Cav1.342A) or RIM2α/RBP2 (Cav1.3L) reduced Cav1.3 VDI to a similar extent as observed in IHCs. Membrane-anchored β2 variants (β2a, β2e) that inhibit inactivation on their own allowed no further modulation of inactivation kinetics by RIM2α/RBP2. Moreover, association with RIM2α and/or RBP2 consolidated the negative Cav1.3 voltage operating range by shifting the channel's activation threshold toward more hyperpolarized potentials. Taken together, the association with "slow" β subunits (β2a, β2e) or presynaptic scaffolding proteins such as RIM2α and RBP2 stabilizes physiological gating properties of IHC Cav1.3 LTCCs in a splice variant-dependent manner ensuring proper IHC function. [ABSTRACT FROM AUTHOR]

In cardiomyocytes, electrical activity is coupled to cellular contraction, thus exposing all proteins expressed in the sarcolemma to mechanical stress. The voltage-gated sodium channel Nav1.5 is the main contributor to the rising phase of the action potential in the heart. There is growing evidence that gating and kinetics of Nav1.5 are modulated by mechanical forces and pathogenic variants that affect mechanosensitivity have been linked to arrhythmias. Recently, the sodium channel β1 subunit has been described to stabilise gating against mechanical stress of Nav1.7 expressed in neurons. Here, we tested the effect of β1 and β3 subunits on mechanosensitivity of the cardiac Nav1.5. β1 amplifies stress-induced shifts of V1/2 of steady-state fast inactivation to hyperpolarised potentials (ΔV1/2: 6.2 mV without and 10.7 mV with β1 co-expression). β3, on the other hand, almost doubles stress-induced speeding of time to sodium current transient peak (Δtime to peak at − 30 mV: 0.19 ms without and 0.37 ms with β3 co-expression). Our findings may indicate that in cardiomyocytes, the interdependence of electrical activity and contraction is used as a means of fine tuning cardiac sodium channel function, allowing quicker but more strongly inactivating sodium currents under conditions of increased mechanical stress. This regulation may help to shorten action potential duration during tachycardia, to prevent re-entry phenomena and thus arrhythmias. [ABSTRACT FROM AUTHOR]

Molecular pain 2:33. https://doi.org/10.1186/1744-8069-2-33 10 Ohtubo Y (2021) Slow recovery from the inactivation of voltage-gated sodium channel Nav1.3 in mouse taste receptor cells. Types II and III directly function as taste receptor cells, but the cascade of steps leading to secretion of signaling molecules from TBCs and stimulation of postsynaptic afferent nerves is entirely different in the two cases [[6]]. Subsequent single-cell PCR that included cell-specific markers revealed a higher frequency of Nav1.3 message among the sets of types II and III TBCs, while the TTX-resistant Nav1.5 was observed in type I TBCs. [Extracted from the article]

Adrenal chromaffin cells (CCs) are the main source of circulating catecholamines (CAs) that regulate the body response to stress. Release of CAs is controlled neurogenically by the activity of preganglionic sympathetic neurons through trains of action potentials (APs). APs in CCs are generated by robust depolarization following the activation of nicotinic and muscarinic receptors that are highly expressed in CCs. Bovine, rat, mouse, and human CCs also express a composite array of Na, K, and Ca channels that regulate the resting potential, shape the APs, and set the frequency of AP trains. AP trains of increasing frequency induce enhanced release of CAs. If the primary role of CCs is simply to relay preganglionic nerve commands to CA secretion, why should they express such a diverse set of ion channels? An answer to this comes from recent observations that, like in neurons, CCs undergo complex firing patterns of APs suggesting the existence of an intrinsic CC excitability (non-neurogenically controlled). Recent work has shown that CCs undergo occasional or persistent burst firing elicited by altered physiological conditions or deletion of pore-regulating auxiliary subunits. In this review, we aim to give a rationale to the role of the many ion channel types regulating CC excitability. We will first describe their functional properties and then analyze how they contribute to pacemaking, AP shape, and burst waveforms. We will also furnish clear indications on missing ion conductances that may be involved in pacemaking and highlight the contribution of the crucial channels involved in burst firing. [ABSTRACT FROM AUTHOR]

The epithelial sodium channel (ENaC) constitutes the rate-limiting step for Na transport across electrically tight epithelia. Regulation of ENaC activity is critical for electrolyte and extracellular volume homeostasis, as well as for lung liquid clearance and colon Na handling. ENaC activity is tightly controlled by a combination of mechanisms involving changes in open probability and plasma membrane abundance. The latter reflects a combination in channel biosynthesis and trafficking to and from the membrane. Studying ENaC trafficking with different techniques in a variety of expression systems has yielded inconsistent results, indicating either fast or slow rates of insertion and retrieval, which range from the order of minutes to several hours. Here, we use Xenopus oocytes as ENaC expression system to study channel insertion rate in the membrane using two different techniques under comparable conditions: (1) confocal microscopy coupled to fluorescence recovery after photobleaching (FRAP) measurements; and (2) fluorescent bungarotoxin (BTX) binding to ENaC subunits modified to include BTX binding sites (BBSs) in their extracellular domain, a technique that has not been previously used to study ENaC trafficking. Our confocal-FRAP data indicate a fast rate of ENaC incorporation to the membrane in a process conditioned by channel subunit composition. On the other hand, BTX binding experiments indicate much slower channel insertion rates, with matching slow ENaC retrieval rates. The data support a model that includes fast recycling of endocytosed ENaC with parallel incorporation of newly synthesized channels at a slower rate. [ABSTRACT FROM AUTHOR]

Solutions of table salt (NaCl) elicit several tastes, including of course saltiness but also sweet, sour, and bitter. This brief review touches on some of the mileposts concerning what is known about taste transduction for the Na ion, the main contributor to saltiness. Electrophysiological recordings, initially from single gustatory nerve fibers, and later, integrated impulse activity from gustatory nerves led researchers to predict that Na ions interacted with a surface molecule. Subsequent studies have resolved that this molecule is likely to be an epithelial sodium channel, ENaC. Other Na transduction mechanisms are also present in taste buds but have not yet been identified. The specific type(s) of taste cells responsible for salt taste also remains unknown. [ABSTRACT FROM AUTHOR]

Tetrodotoxin (TTX) is believed to be the most selective inhibitor of voltage-gated fast Na channels in excitable tissues, including nerve, skeletal muscle, and heart, although TTX sensitivity of the latter is lower than the former by at least three orders of magnitude. In the present study, the TTX sensitivity of L-type Ca current ( I) was studied in isolated canine ventricular cells using conventional voltage clamp and action potential voltage clamp techniques. TTX was found to block I in a reversible manner without altering inactivation kinetics of I. Fitting results to the Hill equation, an IC value of 55 ± 2 μM was obtained with a Hill coefficient of unity (1.0 ± s0.04). The current was fully abolished by 1 μM nisoldipine, indicating that it was really I. Under action potential voltage clamp conditions, the TTX-sensitive current displayed the typical fingerprint of I, which was absent in the presence of nisoldipine. Stick-and-ball models for Cav1.2 and Nav1.5 channel proteins were constructed to explain the differences observed between action of TTX on cardiac I and I. This is the first report demonstrating TTX to interact with L-type calcium current in the heart. [ABSTRACT FROM AUTHOR]

Accentuated right ventricular (RV) gradients in action potential duration (APD) have been implicated in the arrhythmogenicity observed in Brugada syndrome in studies assuming that ventricular effective refractory periods (VERPs) vary in concert with APDs. The present experiments use a genetically modified mouse model to explore spatial heterogeneities in VERP that in turn might affect conduction velocity, thereby causing arrhythmias. Activation latencies, APDs and VERPs recorded during programmed S1S2 protocols were compared in RV and left ventricular (LV) epicardia and endocardia of Langendorff-perfused wild-type (WT) and Scn5a hearts. Scn5a and WT hearts showed similar patterns of shorter VERPs in RV than LV epicardia, and in epicardia than endocardia. However, Scn5a hearts showed longer VERPs, despite shorter APDs, than WT in all regions examined. The pro- and anti-arrhythmic agents flecainide and quinidine increased regional VERPs despite respectively decreasing and increasing the corresponding APDs particularly in Scn5a RV epicardia. In contrast, Scn5a hearts showed greater VERP gradients between neighbouring regions, particularly RV transmural gradients, than WT (9.1 ± 1.1 vs. 5.7 ± 0.5 ms, p < 0.05, n = 12). Flecainide increased (to 21 ± 0.9 ms, p < 0.05, n = 6) but quinidine decreased (to 4.5 ± 0.5 ms, p < 0.05, n = 6) these gradients, particularly across the Scn5a RV. Finally, Scn5a hearts showed greater conduction slowing than WT following S2 stimuli, particularly with flecainide administration. Rather than arrhythmogenesis resulting from increased transmural repolarization gradients in an early, phase 2, reentrant excitation mechanism, the present findings implicate RV VERP gradients in potential reentrant mechanisms involving impulse conduction slowed by partial refractoriness. [ABSTRACT FROM AUTHOR]

Whole-cell, patch-clamp recordings were carried out in acutely dissociated neurons from entorhinal cortex (EC) layer II to study the effects of Zn on Na current kinetics and voltage dependence. In the presence of 200 μM extracellular Cd to abolish voltage-dependent Ca currents, and 100 mM extracellular Na, 1 mM Zn inhibited the transient Na current, I, only to a modest degree (~17% on average). A more pronounced inhibition (~36%) was induced by Zn when extracellular Na was lowered to 40 mM. Zn also proved to modify I voltage-dependent and kinetic properties in multiple ways. Zn (1 mM) shifted the voltage dependence of I activation and that of I onset speed in the positive direction by ~5 mV. The voltage dependence of I steady-state inactivation and that of I inactivation kinetics were markedly less affected by Zn. By contrast, I deactivation speed was prominently accelerated, and its voltage dependence was shifted by a significantly greater amount (~8 mV on average) than that of I activation. In addition, the kinetics of I recovery from inactivation were significantly slowed by Zn. Zn inhibition of I showed no signs of voltage dependence over the explored membrane-voltage window, indicating that the above effects cannot be explained by voltage dependence of Zn-induced channel-pore block. These findings suggest that the multiple, voltage-dependent state transitions that the Na channel undergoes through its activation path are differentially sensitive to the gating-modifying effects of Zn, thus resulting in differential modifications of the macroscopic current's activation, inactivation, and deactivation. Computer modeling provided support to this hypothesis. [ABSTRACT FROM AUTHOR]

Ion channels and transporters play a critical role in ion and fluid homeostasis and thus in normal animal physiology and pathology. Tight regulation of these transmembrane proteins is therefore essential. In recent years, many studies have focused their attention on the role of the ubiquitin system in regulating ion channels and transporters, initialed by the discoveries of the role of this system in processing of Cystic Fibrosis Transmembrane Regulator (CFTR), and in regulating endocytosis of the epithelial Na channel (ENaC) by the Nedd4 family of ubiquitin ligases (mainly Nedd4-2). In this review, we discuss the role of the ubiquitin system in ER Associated Degradation (ERAD) of ion channels, and in the regulation of endocytosis and lysosomal sorting of ion channels and transporters, focusing primarily in mammalian cells. We also briefly discuss the role of ubiquitin like molecules (such as SUMO) in such regulation, for which much less is known so far. [ABSTRACT FROM AUTHOR]

Bacterial lipopolysaccharides (LPS) are potent inducers of proinflammatory signaling pathways via the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK), causing changes in the processes that control lung fluid homeostasis and contributing to the pathogenesis of lung disease. In human H441 airway epithelial cells, incubation of cells with 15 µg ml−1 LPS caused a significant reduction in amiloride-sensitive Isc from 15 ± 2 to 8 ± 2 µA cm−2 ( p = 0.01, n = 13) and a shift in IC50 amiloride of currents from 6.8 × 10−7 to 6.4 × 10−6 M. This effect was associated with a decrease in the activity of 5 pS, highly Na+ selective, amiloride-sensitive <1 µM channels (HSC) and an increase in the activity of ∼18 pS, nonselective, amiloride-sensitive >10 µM cation channels (NSC) in the apical membrane. LPS decreased αENaC mRNA and protein abundance, inferring that LPS inhibited αENaC gene expression. This correlated with the decrease in HSC activity, indicating that these channels, but not NSCs, were comprised of at least αENaC protein. LPS increased NF-κB DNA binding activity and phosphorylation of extracellular signal-related kinase (ERK)1/2, but decreased phosphorylation of ERK5 in H441 cells. Pretreatment of monolayers with PD98059 (20 µM) inhibited ERK1/2 phosphorylation, promoted phosphorylation of ERK5, increased αENaC protein abundance, and reversed the effect of LPS on Isc and the shift in amiloride sensitivity. Inhibitors of NF-κB activation were without effect. Taken together, our data indicate that LPS acts via ERK signaling pathways to decrease αENaC transcription, reducing HSC/ENaC channel abundance, activity, and transepithelial Na+ transport in H441 airway epithelial cells. [ABSTRACT FROM AUTHOR]

Basic principles of the gating mechanisms of neuronal sodium channels, especially the fast inactivation process, were revealed by a quantitative analysis of the effects of the chemically irreversible modifying agent chloramine T. The compound is known to enhance the open probability of sodium channels by interfering with the inactivation process. The key for the deduction of structure–function relationships was obtained from the analysis of single-channel patch-clamp data, especially the finding that chloramine T-induced modification of inactivation occurred in four steps. These steps were termed modes 1–4 (four-mode gating model), and their temporal sequence was always the same. The kinetic analysis of single-channel traces with an improved two-dimensional dwell-time fit revealed the possible mechanism related to each mode. Similarities to the kinetics of the sodium channel mutant F1489Q led to the assignment of modes 1 and 2 to transient defects in the locking of the inactivation particle (hinged lid). In the third mode, the hinged lid was unable to lock permanently. Finally, in mode 4, the apparent single-channel current was reduced, which could be explained by fast gating, presumably related to the selectivity filter. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels play a crucial role in the initiation and propagation of neuronal action potentials. Genistein, an isoflavone phytoestrogen, has long been used as a broad-spectrum inhibitor of protein tyrosine kinases (PTK). In addition, genistein-induced modulation of ion channels has been described previously in the literature. In this study, we investigated the effect of genistein on voltage-gated sodium channels in rat superior cervical ganglia (SCG) neurons. The results show that genistein inhibits Na+ currents in a concentration-dependent manner, with a concentration of half-maximal effect (IC50) at 9.1 ± 0.9 μM. Genistein positively shifted the voltage dependence of activation but did not affect inactivation of the Na+ current. The inactive genistein analog daidzein also inhibited Na+ currents, but was less effective than genistein. The IC50 for daidzein-induced inhibition was 20.7 ± 0.1 μM. Vanadate, an inhibitor of protein tyrosine phosphatases, partially but significantly reversed genistein-induced inhibition of Na+ currents. Other protein tyrosine kinase antagonists such as tyrphostin 23, an erbstatin analog, and PP2 all had small but significant inhibitory effects on Na+ currents. Among all active and inactive tyrosine kinase inhibitors tested, genistein was the most potent inhibitor of Na+ currents. These results suggest that genistein inhibits Na+ currents in rat SCG neurons through two distinct mechanisms: protein tyrosine kinase-independent, and protein tyrosine kinase-dependent mechanisms. Furthermore, the Src kinase family may be involved in the basal phosphorylation of the Na+ channel. [ABSTRACT FROM AUTHOR]

The effects of veratridine have been compared on tetrodotoxin-sensitive (TTXS) and tetrodotoxin-resistant (TTXR) voltage-gated sodium channels (VGSC) in rat dorsal root ganglion neurons. Veratridine caused a dose-dependent decrease in the peak amplitude of both TTXR and TTXS VGSC currents. When exposed to 25 μM veratridine, TTXS currents but not TTXR currents developed a clear persistent component. The deactivation of both TTXS and TTXR currents was slowed, as evidenced by the appearance of slowly decaying tail currents in voltage clamp records, but the slowing of deactivation was nearly 100 times greater for TTXS than for TTXR currents. Properties of the veratridine-modified VGSCs, derived from an analysis of the slow tail currents, were similar for both TTXS and TTXR in that the V50 for activation and the reversal potential were shifted to more negative potentials than control currents and by a similar amount for each. The relatively fast decay of veratridine-modified TTXR tail currents reflects a faster dissociation of veratridine from TTXR than from TTXS VGSCs. This difference probably underlies the lack of effect of veratridine on TTXR VGSCs in cells that are not voltage-clamped and undermines its value as a chemical activator of putative NaV1.8 TTXR channels. [ABSTRACT FROM AUTHOR]

The epithelial sodium channel (ENaC) is the major mediator of sodium transport across the apical membranes of the distal nephron, the distal colon, the respiratory tract and the ducts of exocrine glands. It is subject to feedback inhibition by increased intracellular Na+, a regulatory system wherein the ubiquitin protein ligases, Nedd4 and Nedd4-2, bind to conserved PY motifs in the C-termini of ENaC and inactivate the channel. It has been proposed recently that the kinase Sgk activates the channel as a consequence of phosphorylating Nedd4-2, thus preventing it from inhibiting the channels. This proposal predicts that Sgk should interfere with Na+ feedback regulation of ENaC. We have tested this prediction in Xenopus laevis oocytes and in mouse salivary duct cells and found that in neither system did increased activity of Sgk interrupt Na+ feedback inhibition of ENaC. We found, however, that Sgk stimulation was largely abolished in oocytes expressing ENaC channels with C-terminal truncations or mutated PY motifs. We were also unable to confirm that Sgk directly interacts with Nedd4-2 in vitro. We conclude that the stimulatory effect of Sgk on ENaC requires the presence of the channel’s PY motifs, but it is not due to the interruption of Na+ feedback regulation. [ABSTRACT FROM AUTHOR]

The mode of action of the alkaloid veratridine has been reinvestigated on cultured cockroach neurones, which are normally inexcitable and do not have a detectable fast sodium current. The whole-cell and cell-attached configurations of the patch-clamp technique were used to record the macroscopic and single channel currents, respectively. Concentrations of veratridine ranging from 10 to 10 M were found to induce a small tetrodotoxin (TTX)-sensitive inward current, which peaked around + 10 mV and reversed around + 55 mV. This current exhibited a pronounced plateau and was insensitive to changes in the holding potential. Bath application of veratridine induced typical TTX-sensitive inwardly-directed single-channel activity, falling into two (apparently coupled) categories of events: first, relatively large events (1 pA at a hyperpolarized potential of −125 mV relative to rest) of short duration and, second, small bursting events (0.4 pA under similar conditions) of slightly longer duration. Pipette application of similar concentrations of veratridine had similar effects in that two categories of events were observed: first, bursts of large events with multiple conductance states and, second, small events of very long duration. The current/ voltage relationship of these events was linear for the voltage range studied and the (extrapolated) reversal potential approximated + 110 mV. These results support the hypothesis that veratridine, in small concentrations, induces a slow voltage-dependent activation of TTX-sensitive sodium channels, independent of the fast activating and inactivating sodium channels involved in action potential generation. [ABSTRACT FROM AUTHOR]

Neurones of the neostriatum were freshly dispersed from the adult guinea pig brain. A fast, transient inward Na current ( I) was analysed using the whole-cell patch-clamp technique. Upon depolarizations, I developed with a sigmoidal time course, which was described by m kinetics. I showed an activation threshold of about −60 mV, a peak current at −30 to −20 mV, and a reversal of polarity at +60 mV. The steady-state activation (m) curve for I had a slope factor of about 9 mV with a mid-point potential of about −26 mV. The voltage dependence of the activation time constant, τ, had a bell-shaped configuration with a maximum value at −60 mV. The forward rate constant for I activation (α) increased as the membrane was depolarized (about 9 mV for a change in the rate constant by a factor of e) in the range between −50 mV and −20 mV. Conversely, the backward rate constant ( β) decreased as the membrane was depolarized (about 31 mV for an e-fold change). The steady-state inactivation ( h) curve was well expressed by the Boltzmann's equation with a half-inactivation potential of −62 mV and a slope parameter of 6 mV. The time course of I decay followed a second-order process, whereas the recovery from inactivation was described as a first-order process. The τ curve showed a bell-shaped configuration with a maximum value at −60 mV. The forward rate constants (α) decreased as the membrane was depolarized (about 17 mV for an e-fold change) in the range between −50 mV and −20 mV. The backward rate constants ( β) increased as the membrane was depolarized (about 10 mV for an e-fold change). There was a significant overlap between m and h curves, suggesting a steady influx of Na (window current). [ABSTRACT FROM AUTHOR]

The effects of external divalent cations on the steady-state open probability ( P), opening and closing rates, and conductance of batrachotoxin (BTX)-activated Na channels were studied in planar lipid bilayers. External divalent cations shifted the midpoint of the P versus membrane potential ( V) relation (gating curve) to more depolarized potentials. Of the group IIA divalent cations tested, Ca caused the largest depolarizing shift followed by Ba, Sr and Mg in that order. In contrast, the order of decreasing efficacy for block of the single channel conductance was Ca, Mg, Sr and Ba. Thus, because it is a relatively good shifter but a poor blocker, Ba was used to study the effects of divalent cations on gating. External Ba decreased the Na channel opening rate and increased the closing rate. At voltages close to the midpoint of the gating curve, the opening rate decreased by a factor of 1/2.8 while the closing rate increased by 2.0-fold. Although Ba would be expected to increase the external surface potential by interacting with negatively charged residues on the channel, this preferential effect on the opening rate indicates that the effects of external Ba cannot be attributed solely to an increase in the potential gradient across the voltage sensing apparatus. Our results can be explained if two different mechanisms are involved in the modulation of Na channel gating by extracellular divalent cations in neutral membranes: (1) the screening or binding of Ba at negatively charged groups on the extracellular side of the channel, leading to an increase in the transmembrane electric field at the voltage sensor, as well as an increase in the closing rate and decrease in the opening rate by the same proportion, plus (2) a direct modulatory action on the channel protein which stabilizes the closed state of the channel and accounts for the larger effect of external divalent cations on the opening rate. [ABSTRACT FROM AUTHOR]

The effects of lysophosphatidylcholine (LPC) on Na channels in inside-out patches of adult rat ventricular cells using the patch-clamp technique have been investigated. Application of LPC (9-25 μM) from the inner side of membrane for 4-15 min caused a reduction of averaged Na current (I) peak and prolonged the time course of inactivation in the potential range of -50 to -10 mV. Analysis of single channel behaviour revealed that after 30-50 min of exposure, in addition to normally functioning Na channels with short openings, LPC induced longlasting bursts of Na channel openings (up to the 300 ms duration of the test pulses). This resulted in an appearance of noninactivated component of I. The slope conductance of these modified channels remained the same as in control (11.3 pS - control; 11.6 pS - LPC-treated). The dwell time for modified channels increased significantly. [ABSTRACT FROM AUTHOR]