Cell volume analysis of gramicidin-treated eccrine clear cells to study regulation of Cl channels

Using voltage-current-clamp methods, we determined membrane potentials, relative ionic permeability, and membrane conductance of gramicidin (GC)-treated freshly dissociated eccrine clear cells. GC depolarized the membrane potential by 58 mV, increased the membrane conductance progressively over the time of exposure (mean of 1.7 times at 60 s and 4.6 times at 3 min), and increased the Na conductance of the membrane (from near 0 in control to 0.75 nS after GC). Image analysis coupled with GC treatment was then employed to study the regulation of Cl channels based on the premise that cell swelling was due to activation of Cl channels. Cell swelling was stimulated by methacholine (MCh, 3 microM) in the presence of GC. GC+MCh-induced cell swelling was inhibited by atropine, low extracellular Ca ([Ca]o < 1 nM), or removal of Cl. Thus MCh-induced cell swelling is most likely due to Ca-dependent activation of Cl channels. Isoproterenol (Iso), 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, 3-isobutyl-1-methylxanthine, and forskolin also caused cell swelling in the presence of GC. Iso-induced cell swelling was abolished in a Cl-free medium and by diphenylamine-2-carboxylic acid, indicating that it is caused by adenosine 3',5'-cyclic monophosphate (cAMP)-mediated activation of Cl channels. Cl channels stimulated by MCh, but not those stimulated by Iso, were inhibited by preexposure to a low-Ca medium [nominally Ca free + 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, [Ca]o < 1 nM] for 20 s, suggesting that Ca-stimulated Cl channels are distinct from cAMP-dependent Cl channels. cAMP-stimulated Cl channels were, however, inhibited when the cells were exposed to the low-Ca medium for 60 s. The simple cell volume analysis of GC-treated cells is a sensitive assay system for both Ca- and cAMP-dependent Cl channels.(ABSTRACT TRUNCATED AT 250 WORDS)