Abstract
Cytoskeletal elements play an important role in the regulation of ion transport in epithelia. We have studied the effects of actin filaments of different length on the alpha, beta, gamma-rENaC (rat epithelial Na+ channel) in planar lipid bilayers. We found the following. 1) Short actin filaments caused a 2-fold decrease in unitary conductance and a 2-fold increase in open probability (Po) of alpha,beta,gamma-rENaC. 2) alpha,beta,gamma-rENaC could be transiently activated by protein kinase A (PKA) plus ATP in the presence, but not in the absence, of actin. 3) ATP in the presence of actin was also able to induce a transitory activation of alpha, beta,gamma-rENaC, although with a shortened time course and with a lower magnitude of change in Po. 4) DNase I, an agent known to prohibit elongation of actin filaments, prevented activation of alpha,beta,gamma-rENaC by ATP or PKA plus ATP. 5) Cytochalasin D, added after rundown of alpha,beta,gamma-rENaC activity following ATP or PKA plus ATP treatment, produced a second transient activation of alpha,beta,gamma-rENaC. 6) Gelsolin, a protein that stabilizes polymerization of actin filaments at certain lengths, evoked a sustained activation of alpha,beta,gamma-rENaC at actin/gelsolin ratios of <32:1, with a maximal effect at an actin/gelsolin ratio of 2:1. These results suggest that short actin filaments activate alpha, beta,gamma-rENaC. PKA-mediated phosphorylation augments activation of this channel by decreasing the rate of elongation of actin filaments. These results are consistent with the hypothesis that cloned alpha,beta,gamma-rENaCs form a core conduction unit of epithelial Na+ channels and that interaction of these channels with other associated proteins, such as short actin filaments, confers regulation to channel activity.
Highlights
They showed, in patch-clamp experiments of ratio of 2:1. These results suggest that short actin fila- apical membranes of A6 epithelial cells grown on glass coverments activate ␣,,␥-rat epithelial Naϩ channel (rENaC)
The actin cytoskeleton has been shown to interact with a variety of transmembrane proteins, including ion transport molecules such as the band 3 anion exchanger [9], the epithelial Naϩ/Kϩ-ATPase [6, 10], rat brain undertaken to test the hypothesis that short actin filaments could modulate the activity of rENaC and that PKA sensitivity could be conferred onto rENaC, but only in the presence of actin
Effect of Actin on Single ␣,,␥-rENaC—The interaction of epithelial Naϩ channels with cytoskeletal proteins in native epithelia prompted us to examine the hypothesis that the activity of cloned ␣,,␥-rENaC may be modulated by actin
Summary
Cantiello and co-workers [15, 24] have provided strong evidence that the actin component of the membrane cytoskeleton is involved in the regulation of A6 renal epithelial Naϩ chantios of
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