Triple-barrel Organization of ENaC, a Cloned Epithelial Na+ Channel

摘要: A cloned rat epithelial Na+ channel (rENaC) was studied in planar lipid bilayers. Two forms of the were examined: channels produced by alpha subunit alone and those formed alpha, beta, gamma subunits. The protein derived from two sources: either vitro translation reaction followed Sephadex column purification or heterologous expression Xenopus oocytes isolation plasma membranes. We found that alpha-rENaC alpha- combination with beta- gamma-rENaC, highly Na(+)-selective (PNa/PK = 10), amiloride-sensitive (Kamili 170 nM), mechanosensitive cation displayed a complicated gating mechanism: there nearly constitutively open 13-picosiemens (pS) state second 40-pS level achieved 13-pS 26-pS transition. alpha-, beta-, gamma-rENaC showed primarily level. alpha,beta,gamma-rENaC patch clamp same pattern, albeit > 2-fold lowered conductance levels, i.e. 6 18 pS, respectively. Upon treatment sulfhydryl reducing agent dithiothreitol, both fluctuated among three independent sublevels. Bathing each high salt solution (1.5 M NaCl) stochastic openings 19 38 pS magnitude between all levels. Different combinations reconstitution mixture did not produce intermediate These findings suggest functional ENaC is composed identical conducting elements their concerted.