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

1. A weak electroneutral sodium channel blocker 6-chloro-3,5-diamino-pyrazine-2-carboxamide was used to perform noise analysis on isolated epithelium from Rana fuscigula to determine the cellular mechanism underlying autoregulation of Na+ channel densities in response to a reduction in the mucosal Na+ concentration. 2. The inherent transport rates of these tissues were generally lower than in other frog skins. The macroscopic sodium current, INa, averaged 10.71 microA/cm2 and was mainly determined by the number of open channels (N(o)) which averaged 21.6 million/cm2. The calculated mean channel open probability (beta') was 0.38, and corresponded very closely to values previously determined by patch clamp. 3. Reducing the mucosal Na+ from 110 to 10 mM caused large increases in the open channel density, which stabilized the Na+ transport rate. N(o) increased from a mean value of 26.6 to 64.3 million/cm2 within 2 min. 4. Autoregulatory changes were induced primarily by increasing beta' by about 60% and to a lesser extent by an increase in NT, the total number of open and closed channels. 5. We also examined the role of the cytoskeleton in the regulation of Na+ channel densities. Colchicine treatment, which disrupted microtubules, had no apparent effect on the ability of the tissues to autoregulate their Na+ channel densities. 6. The integrity of the microfilaments were essential for autoregulatory changes in N(o). After we had disrupted the microfilaments with cytochalasin B, we observed a marked reduction in the ability of the tissues to increase N(o). 7. The mean N(o) did not increase in response to a drop in mucosal Na+ despite the fact that beta' increased by 69%. We, therefore, assumed that cytochalasin B did not affect Na+ channels already present in the membrane but interfered with recruitment of new channels. Significantly, we did not observe any increase in NT. 8. In kidney and other tight epithelia, microfilaments are responsible for regulating the delivery of newly synthesized membrane proteins. We believe that our results with cytochalasin-treated tissues support the theory that autoregulatory changes in N(o) are also regulated by the recruitment of channels from a cytoplasmic pool.