IN-VITRO PATCH-CLAMP STUDIES IN SKIN FIBROBLASTS

We have conducted single-channel patch-clamp experiments in skin fibro blasts maintained in culture. Two different cell lines, a mouse 3T3-L1 cell line and a human B17 cell line, were selected for these pilot st udies. Recordings were made from both cell-attached and excised inside -out patches at room temperature. In the case of the 3T3-L1 cells, the success rate in obtaining good seals (> 1G Ohm) was low, and channel openings in either cell-attached or excised patches were rare. We have , however, identified a channel in a cell-attached configuration with a slope conductance of 39 pS in symmetrical K+ solutions. In the case of the human B17 cells, good quality seals were more readily obtained. One principal type of channel opening was identified. In cell-attache d patches, the prevalent type of channel in symmetrical K+ solutions h ad a conductance of 187 pS. This channel was activated by strong depol arization, and there was usually more than one active channel in the p atch. It was blocked by extracellular tetraethylammonium (20 mM), and persisted when external Cl- was replaced by aspartate. In excised insi de-out patches bathed in symmetrical K+, this channel was activated by an increase in Ca+ applied to the intracellular face. A large conduct ance channel (175 pS) was also observed in excised inside-out patches, with a reverse physiological K+ gradient. This channel had a reversal potential > 40 mV and appeared not to be voltage-dependent under thes e recording conditions (2 mM Ca-i(2+ )). We conclude that the channel we have identified in these cells belongs to the maxi-K+ channel class . (C) 1998 Elsevier Science Inc.