On mutations that uncouple sodium channel activation from inactivation.
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Computations on sodium channel gating were conducted using a closed-open-inactivated coupled kinetic scheme. The time constant of inactivation (tauh) derives a voltage dependency from coupling to voltage-dependent activation even when rate constants between inactivated and other states are strictly voltage independent. The derived voltage dependency does not require any physical, molecular link between the structures responsible for inactivation and the charges producing voltage-dependent activation. The only requirement is that the closed to inactivated rate constant (kCI) differs from the open to inactivated (kOI), consistent with experimental results. A number of mutations and other treatments uncouple sodium channel activation and inactivation in that the voltage dependency of tauh is substantially reduced while voltage-dependent activation persists. However, a clear basis for uncoupling has not been described. A variety of experimental results are accounted for just by changes in the difference between kOI and kCI. In wild type channels, kOI > kCI and inactivation develops with a delay whose time constant is just that for channel opening. Mutations that reduce the kOI - kCI difference reduce the amplitude of the delay process and the derived voltage dependency of tauh. If kOI = kCI, inactivation develops as a single exponential (no matter what the number of closed states), activation and inactivation become independent, parallel processes, and any voltage dependency of tauh is then entirely intrinsic to inactivation. If kOI < kCI, inactivation develops as the sum of exponentials, tauh at negative potentials speeds and then slows with more positive potentials. These predicted kOI < kCI effects have all been seen experimentally (O'Leary, M.E., L.-Q. Chen, R.G. Kallen, and R. Horn. 1995. J. Gen. Physiol. 106: 641-658). An open to closed rate constant of zero also removes the derived voltage dependency of tauh, but activation and inactivation are still coupled and the inactivation delay remains. (+info)
Epithelial Na+ channels (ENaC) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) upon activation by protein kinase A. It is, however, still unclear how CFTR regulates the activity of ENaC. In the present study we examined whether CFTR interacts with ENaC by interfering with the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various C-terminal mutations were introduced into the three alpha-, beta-, and gamma-subunits of the rat epithelial Na+ channel, thereby eliminating PY motifs, which are important binding domains for the ubiquitin ligase Nedd4. When expressed in Xenopus oocytes, most of the ENaC stop (alpha-H647X, beta-P565X, gamma-S608X) or point (alpha-P671A, beta-Y618A, gamma-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type alpha,beta,gamma-rENaC. However, ENaC currents formed by either of the mutant alpha-, beta-, or gamma-subunits were inhibited during activation of CFTR by forskolin (10 micromol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced alpha,beta,gamma-rENaC whole cell Na+ conductance but did not interfere with inhibition of ENaC by CFTR. Another mutant, beta-T592M,T593A-ENaC, also showed enhanced Na+ currents, which were down-regulated by CFTR. Moreover, activation of ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that regulation of ENaC by CFTR is distal to other regulatory limbs and does not involve Nedd4-dependent ubiquitination. (+info)
Enhancing effects of salicylate on tonic and phasic block of Na+ channels by class 1 antiarrhythmic agents in the ventricular myocytes and the guinea pig papillary muscle.
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OBJECTIVE: To study the interaction between salicylate and class 1 antiarrhythmic agents. METHODS: The effects of salicylate on class 1 antiarrhythmic agent-induced tonic and phasic block of the Na+ current (INa) of ventricular myocytes and the upstroke velocity of the action potential (Vmax) of papillary muscles were examined by both the patch clamp technique and conventional microelectrode techniques. RESULTS: Salicylate enhanced quinidine-induced tonic and phasic block of INa at a holding potential of -100 mV but not at a holding potential of -140 mV; this enhancement was accompanied by a shift of the hinfinity curve in the presence of quinidine in a further hyperpolarized direction, although salicylate alone did not affect INa. Salicylate enhanced the tonic and phasic block of Vmax induced by quinidine, aprindine and disopyramide but had little effect on that induced by procainamide or mexiletine; the enhancing effects were related to the liposolubility of the drugs. CONCLUSIONS: Salicylate enhanced tonic and phasic block of Na+ channels induced by class 1 highly liposoluble antiarrhythmic agents. Based on the modulated receptor hypothesis, it is probable that this enhancement was mediated by an increase in the affinity of Na+ channel blockers with high lipid solubility to the inactivated state channels. (+info)
BIIR 561 CL: a novel combined antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and voltage-dependent sodium channels with anticonvulsive and neuroprotective properties.
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Antagonists of glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype, as well as of voltage-gated sodium channels, exhibit anticonvulsive and neuroprotective properties in vivo. One can postulate that a compound that combines both principles might be useful for the treatment of disorders of the central nervous system, like focal or global ischemia. Here, we present data on the effects of dimethyl-(2-[2-(3-phenyl-[1,2, 4]oxadiazol-5-yl)-phenoxy]ethyl)-amine hydrochloride (BIIR 561 CL) on neuronal AMPA receptors and voltage-dependent sodium channels. BIIR 561 CL inhibited AMPA receptor-mediated membrane currents in cultured cortical neurons with an IC50 value of 8.5 microM. The inhibition was noncompetitive. In a cortical wedge preparation, BIIR 561 CL reduced AMPA-induced depolarizations with an IC50 value of 10.8 microM. In addition to the effects on the glutamatergic system, BIIR 561 CL inhibited binding of radiolabeled batrachotoxin to rat brain synaptosomal membranes with a Ki value of 1.2 microM. The compound reduced sodium currents in voltage-clamped cortical neurons with an IC50 value of 5.2 microM and inhibited the veratridine-induced release of glutamate from rat brain slices with an IC50 value of 2.3 microM. Thus, BIIR 561 CL inhibited AMPA receptors and voltage-gated sodium channels in a variety of preparations. BIIR 561 CL suppressed tonic seizures in a maximum electroshock model in mice with an ED50 value of 2.8 mg/kg after s.c. administration. In a model of focal ischemia in mice, i.p. administration of 6 or 60 mg/kg BIIR 561 CL reduced the area of the infarcted cortical surface. These data show that BIIR 561 CL is a combined antagonist of AMPA receptors and voltage-gated sodium channels with promising anticonvulsive and neuroprotective properties. (+info)
Malignant human gliomas express an amiloride-sensitive Na+ conductance.
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Human astrocytoma cells were studied using whole cell patch-clamp recording. An inward, amiloride-sensitive Na+ current was identified in four continuous cell lines originally derived from human glioblastoma cells (CH235, CRT, SKMG-1, and U251-MG) and in three primary cultures of cells obtained from glioblastoma multiforme tumors (up to 4 passages). In addition, cells freshly isolated from a resected medulloblastoma tumor displayed this same characteristic inward current. In contrast, amiloride-sensitive currents were not observed in normal human astrocytes, low-grade astrocytomas, or juvenile pilocytic astrocytomas. The only amiloride-sensitive Na+ channels thus far molecularly identified in brain are the brain Na+ channels (BNaCs). RT-PCR analyses demonstrated the presence of mRNA for either BNaC1 or BNaC2 in these tumors and in normal astrocytes. These results indicate that the functional expression of amiloride-sensitive Na+ currents is a characteristic feature of malignant brain tumor cells and that this pathway may be a potentially useful target for therapeutic intervention. (+info)
Sodium action potentials are not required for light-evoked release of GABA or glycine from retinal amacrine cells.
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Although most CNS neurons require sodium action potentials (Na-APs) for normal stimulus-evoked release of classical neurotransmitters, many types of retinal and other sensory neurons instead use only graded potentials for neurotransmitter release. The physiological properties and information processing capacity of Na-AP-producing neurons appear significantly different from those of graded potential neurons. To classify amacrine cells in this dichotomy, we investigated whether Na-APs, which are often observed in these cells, are required for functional light-evoked release of inhibitory neurotransmitters from these cells. We recorded light-evoked inhibitory postsynaptic currents (IPSCs) from retinal ganglion cells, neurons directly postsynaptic to amacrine cells, and applied TTX to block Na-APs. In control solution, TTX application always led to partial suppression of the light-evoked IPSC. To isolate release from glycinergic amacrine cells, we used either bicuculline, a GABAA receptor antagonist, or picrotoxin, a GABAA and GABAC receptor antagonist. TTX application only partially suppressed the glycinergic IPSC. To isolate release from GABAergic amacrine cells, we used the glycine receptor blocker strychnine. TTX application only partially suppressed the light-evoked GABAergic IPSC. Glycinergic and GABAergic amacrine cells did not obviously differ in the usage of Na-APs for release. These observations, in conjunction with previous studies of other retinal neurons, indicate that amacrine cells, taken as a class, are the only type of retinal neuron that uses both Na-AP-dependent and -independent modes for light-evoked release of neurotransmitters. These results also provide evidence for another parallel between the properties of retinal amacrine cells and olfactory bulb granule cells. (+info)
Molecular analysis of the Na+ channel blocking actions of the novel class I anti-arrhythmic agent RSD 921.
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RSD 921 is a novel, structurally unique, class I Na+ channel blocking drug under development as a local anaesthetic agent and possibly for the treatment of cardiac arrhythmias. The effects of RSD 921 on wild-type heart, skeletal muscle, neuronal and non-inactivating IFMQ3 mutant neuronal Na+ channels expressed in Xenopus laevis oocytes were examined using a two-electrode voltage clamp. RSD 921 produced similarly potent tonic block of all three wild-type channel isoforms, with EC50 values between 35 and 47 microM, whereas the EC50 for block of the IFMQ3 mutant channel was 110+5.5 microM. Block of Na+ channels by RSD 921 was concentration and use-dependent, with marked frequency-dependent block of heart channels and mild frequency-dependent block of skeletal muscle, wild-type neuronal and IFMQ3 mutant channels. RSD 921 produced a minimal hyperpolarizing shift in the steady-state voltage-dependence of inactivation of all three wild-type channel isoforms. Open channel block of the IFMQ3 mutant channel was best fit with a first order blocking scheme with k(on) equal to 0.11+/-0.012x10(6) M(-1) s(-1) and k(off) equal to 12.5+/-2.5 s(-1), resulting in KD of 117+/-31 microM. Recovery from open channel block occurred with a time constant of 14+/-2.7 s(-1). These results suggest that RSD 921 preferentially interacts with the open state of the Na+ channel, and that the drug may produce potent local anaesthetic or anti-arrhythmic action under conditions of shortened action potentials, such as during anoxia or ischaemia. (+info)
A human muscle Na+ channel mutation in the voltage sensor IV/S4 affects channel block by the pentapeptide KIFMK.
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1. Whole cell patch clamping of transfected HEK293 cells was used to examine the effects of a pentapeptide (KIFMK) containing the proposed inactivation particle of the Na+ channel on two mutations causing myotonia. One mutation (R1448P) is located in the voltage sensor IV/S4, and the other one (G1306E) near the postulated inactivation gate within the III-IV linker. 2. In the absence of peptide, currents of wild-type (WT) and mutant human muscle Na+ channels decayed monoexponentially with inactivation time constants that were 5-fold (R1448P) and 3-fold (G1306E) larger for the mutants. Upon intracellular application of KIFMK (0.3-1 mM) the current decay became biexponential with an additional fast decaying component that increased in amplitude with depolarization. 3. Furthermore, the peptide induced large tail currents upon repolarization, indicating that KIFMK prevents inactivation by blocking open Na+ channels. The peak of this tail current decreased only slowly with depolarizations of increasing duration. The voltage dependence of this decline indicated that the dissociation rate of the charged peptide decreased with depolarization. Increased external [Na+] ([Na+]e) antagonized block by KIFMK, consistent with a pore-blocking mechanism. 4. The results are discussed with regard to a three-state model for one open, an absorbing inactivated and one blocked state with voltage-dependent on- and off-rates for peptide binding. The peptide had qualitatively similar effects on WT and both mutants, indicating that the freely diffusible peptide accelerates the current decay in all three clones. However, for the R1448P mutation the affinity for KFIMK was decreased and the voltage dependence of peptide block was changed in a similar way to the voltage dependence of inactivation. These data suggest that the mutation R1448P affects the voltage-dependent formation of a receptor site for both the inactivation particle and KIFMK. (+info)