The pH sensitivity of a cloned rat kidney K+ channel, ROMK1, was examined after expression in Xenopus oocytes. Membrane currents and intracellular pH (pHi) were concomitantly monitored by the two-microelectrode voltage-clamp technique and a pH-sensitive microelectrode. Oocytes injected with ROMK1 cRNA developed a hyperpolarized resting potential of -98.7 +/- 0.98 mV and a slightly inwardly rectifying Ba(2+)-sensitive K+ current. Lowering external pH from 7.4 to 6.7 using membrane-permeable acetate buffer reduced measured pHi from 7.2 to 6.6 and reduced the ROMK1 current by 80%. The H+ blockade of ROMK1 currents was voltage independent. The relationship between ROMK1 slope conductance and pHi fitted to a titration curve suggested binding of four H+ to a site with a pK of 6.79. Extracellular acidification from pH 7.4 to 6.0 using membrane-impermeable biphthalate buffer had no effect on the ROMK1 current. The pH sensitivity of the ROMK1 channel is similar to that reported for a small-conductance native kidney K+ channel.

No abstract

No abstract

A combination voltage clamp and admittance analysis of single skeletal muscle fibers showed that moderate depolarizations activated a steady-state negative sodium conductance in both the surface and transverse tubular membranes. The density of the voltage-dependent channels was similar for the surface and tubular conductances. The relaxation times associated with the negative conductance were in the millisecond range and markedly potential dependent. The negative tubular conductance has the consequence of increasing the apparent steady-state radial space constant to large values. This occurs because the positive conductance is counterbalanced by the maintained inward-going sodium current. The enhancement of the space constant by a negative conductance provides a means for the nearly simultaneous activation of excitation-contraction coupling.

Early onset effects of methylmercury (MeHg) on recombinant α1β2γ2S or α6β2γ2S subunit-containing GABAA receptors were examined. These are two of the most prevalent receptor types found in cerebellum–a consistent target of MeHg-induced neurotoxicity. Heterologously expressed receptors were used in order to: 1) isolate receptor-mediated events from extraneous effects of MeHg due to stimulation of the receptor secondary to increased release of GABA seen with MeHg in neurons in situ and 2) limit the phenotypes of GABAA receptors present at one time. Initial changes in IGABA in Xenopus laevis oocytes expressing either α1β2γ2S or α6β2γ2S receptors were compared during continuous bath application of MeHg. A time-dependent increase in IGABA mediated by both receptor subtypes occurred following the first 25–30 min of MeHg (5 μM) exposure. In α6β2γ2S containing receptors, the MeHg-induced increase in IGABA was less pronounced compared to that mediated by α1β2γ2S containing receptors, although the pattern of effects was generally similar. Washing with MeHg-free solution reversed the increase in current amplitude. Application of bicuculline at the time of peak potentiation of IGABA rapidly and completely reversed the MeHg-induced currents. Therefore these MeHg-increased inward currents are mediated specifically by the two subtypes of GABAA receptors and appear to entail direct actions of MeHg on the receptor. However bicuculline did not affect stimulation by MeHg of oocyte endogenous Cl−-mediated current, which presumably results from increased [Ca2+]i. Thus, MeHg initially potentiates IGABA in oocytes expressing either α1β2γ2S or α6β2γ2S receptors prior to its more defined later effects, suggesting that MeHg may initially interact directly with GABAA receptors in a reversible manner to cause this potentiation.