Blocker-related changes of channel density. Analysis of a three-state model for apical Na channels of frog skin.

Abstract

Blocker-induced noise analysis of apical membrane Na channels of epithelia of frog skin was carried out with the electroneutral blocker (CDPC, 6-chloro-3,5-diamino-pyrazine-2-carboxamide) that permitted determination of the changes of single-channel Na currents and channel densities with minimal inhibition of the macroscopic rates of Na transport (Baxendale, L. M., and S. I. Helman.1986. Biophys. J. 49:160a). Experiments were designed to resolve changes of channel densities due to mass law action (and hence the kinetic scheme of blocker interaction with the Na channel) and to autoregulation of Na channel densities that occur as a consequence of inhibition of Na transport. Mass law action changes of channel densities conformed to a kinetic scheme of closed, open, and blocked states where blocker interacts predominantly if not solely with open channels. Such behavior was best observed in "pulse" protocol experiments that minimized the time of exposure to blocker and thus minimized the contribution of much longer time constant autoregulatory influences on channel densities. Analysis of data derived from pulse, staircase, and other experimental protocols using both CDPC and amiloride as noise-inducing blockers and interpreted within the context of a three-state model revealed that Na channel open probability in the absence of blocker averaged near 0.5 with a wide range among tissues between 0.1 and 0.9.

Sodium‐dependent regulation of epithelial sodium channel densities in frog skin; a role for the cytoskeleton.

“…In agreement, iNa averaged 0 59 pA, a value comparable to previous results with patch clamp and noise analysis (Helman & Baxendale, 1990;Helman & Kizer, 1990). We calculated that, assuming a mean apical membrane voltage of 85 mV and intracellular Na+ concentration of 14-5 mm (Els & Helman, 1991), singlechannel conductance would be about 4-3 pS.…”

“…The short-circuit current (ISC) and Gt were allowed to reach steady-state levels during control and experimental periods before performing nioise analysis according to the staircase protocol described by Helman & Baxendale (1990). MIicroscopic fluctuations in ISC were induced by sequential introduction of 10 to 50/M of the sodium channel blocker, CDPC (6-chloro-3,5-diamino-pyrazine-2-carboxamide), into the mucosal solution.…”

“…The slow component for the relaxation in ISc could be fitted by a single exponential, typically with time constants of about 9 min for tissues exposed to 20 mm Na+. After 20 min recovery, levels of ISc averaged about 24 % or 35 % below original levels in 20 and 10 mm Na+ respectively ( and hence, analyses were done under conditions close to spontaneous rates of apical Na+ entry (Helman & Baxendale, 1990 ,ctA/cm2, significantly lower than mean values for frog skin from many other species under similar conditions (see Helnian & Kizer, 1990). Often skins with low steady-state control currents were unsuited for our experiments, since experimental conditions and time-dependent transients reduced theISc to such low values (1 or 2 ,uA/cm2) that fluctuation analysis becamne unreliable.…”

“…The use of potent channel blockers in some previous investigations may give cause for concern, specifically with regard to difficulties in measuring off-rate coefficients and determining autoregulatory changes in channel densities (Helman, Cox & Van Driessche, 1983;Abramcheck, Van Driessche & Helman, 1985). We performed noise analysis with the relatively weak electroneutral Na+ channel blocker CDPC (6-chloro-3,5-diamino-pyrazine-2-carboxamide) which provided excellent noise signals at concentrations in a range well below its equilibrium blocker coefficient KB (Helman & Baxendale, 1990), allowing analysis on tissues transporting Na+ near their spontaneous rates. These conditions were particularly conducive to determining single-channel currents, channel densities and, more pertinently, also channel open probabilities (Helman & Baxendale, 1990).…”

“…We performed noise analysis with the relatively weak electroneutral Na+ channel blocker CDPC (6-chloro-3,5-diamino-pyrazine-2-carboxamide) which provided excellent noise signals at concentrations in a range well below its equilibrium blocker coefficient KB (Helman & Baxendale, 1990), allowing analysis on tissues transporting Na+ near their spontaneous rates. These conditions were particularly conducive to determining single-channel currents, channel densities and, more pertinently, also channel open probabilities (Helman & Baxendale, 1990). Our results with noise analysis confirmed a recent finding by Ling & Eaton (1989) with patch clamp that a reduction in mucosal Na+ causes an increase in channel open probability.…”