SUMMARY1. The vaseline-gap voltage-clamp method has been applied to the study of ionic currents in cut pieces from innervated and 5-7 day denervated rat skeletal muscle fibres.2. Kinetic analysis of sodium currents in innervated rat muscle showed them to be similar to those in frog muscle, except that rat sodium channels are activated at slightly more negative potentials. Peak sodium conductances of 40-50 m-mho/cm2 were measured, corresponding to values of GNa of 100-120 m-mho/cm2.3. The permeability sequence of the sodium channel to several organic and inorganic cations is Li+ > Na+ > hydroxylammonium > hydrazinium > guanidiniumã mmonium > K+. TMA+ and Ca2+ were not measurably permeant.4. Denervation appears to shift activation and inactivation parameters of sodium currents by 10 mV to more negative potentials, but does not appreciably affect the maximum peak sodium conductance or the time constants for activation and inactivation.5. Dose-response curves for block by tetrodotoxin in innervated fibres are fitted well by assuming binding of toxin to a single population of channels with a dissociation constant of about 5 nm. In denervated fibres there appears in addition a second population of channels with a dissociation constant in the micromolar range. These relatively toxin-insensitive channels respond less rapidly to potential changes, and can contribute up to 25-30 0% of the total sodium conductance.6. The potassium currents of innervated rat muscle were similar to those of frog muscle in their voltage dependence of activation.7. The time constant for inactivation of the sodium current, Th, at -13 mV showed a temperature dependence measured between 10 and 20 'C equivalent to an average Q10 of 2*3. The Q1o for the time constant of activation of the potassium current, Tn, averaged 2-5 between -40 mV and + 40 mV, measured over the same temperature range.

Abstract-Shear stress-induced activation of endothelial ion channels, one of the earliest responses to flow, is implicated in mechano-signal transduction that results in the regulation of vascular tone. The effects of laminar flow on endothelial membrane potential were studied in vitro using both fluorescent potentiometric dye measurements and whole-cell patch-clamp recordings. The application of flow stimulated membrane hyperpolarization, which was reversed to depolarization within 35 to 160 seconds. The depolarization was caused by a Cl Ϫ -selective membrane current activated by flow independently of the K ϩ channel-mediated hyperpolarization. Thus, flow activated both K ϩ and Cl Ϫ currents, with the net membrane potential being determined by the balance of the responses. Membrane potential sensitivity to flow was unchanged by flow preconditioning that elongated and aligned the cells. (Circ Res. 1999;85:820-828.)

Site-directed mutagenesis was used to mutate alpha Cys418 and beta Cys447 in the M4 domain of Torpedo californica acetylcholine receptor expressed in Xenopus laevis oocytes. The M4 region is a transmembrane domain thought to be located at the lipid-protein interface. By whole-cell voltage clamp analysis, mutation of both alpha subunits to alpha Trp418 increased maximal channel activity approximately threefold, increased the desensitization rate compared with wild-type receptor, and shifted the EC50 for acetylcholine from 32 microM to 13 microM. Patch measurements of single-channel currents revealed that the alpha Trp418 increased channel open times approximately 28-fold at 13 degrees C with no effect on channel conductance. All of our measured functional changes in the alpha Trp418 mutant are consistent with a simple kinetic model of the acetylcholine receptor in which only the channel closing rate is altered by the mutation. Our results show that changes in protein structure at the putative lipid-protein interface can dramatically affect receptor function.

We measured intracellular calcium concentration ([Ca2+]i) during adrenergic stimulation using fura-2 ratio imaging of individual cultured neonatal rat brown fat cells. One micromolar norepinephrine (NE) increased [Ca2+]i from an average resting value of 105 nM to 555 nM in approximately 30 s. [Ca2+]i remained elevated as long as NE was present but returned to resting levels within 2-3 min after NE removal. The response was half maximal at approximately 50 nM NE and was primarily alpha-adrenergic. The sustained, but not the initial, increase in [Ca2+]i required extracellular calcium. Cells stimulated in high-K media had [Ca2+]i responses like those in 0 Ca2+, suggesting that depolarization abrogates calcium influx. Parallel perforated-patch recordings showed that the increase in [Ca2+]i activates a calcium-activated K conductance. Blocking K channels with moderate concentrations of tetraethylammonium (TEA) had only small effects on NE-induced changes in [Ca2+]i, but high concentrations of TEA significantly reduced the response. We conclude that cytoplasmic calcium is modulated by fluxes from both intracellular and extracellular sources and that K channels may not be required for normal short-term [Ca2+]i responses to hormone.