In vitro patch-clamp studies in skin fibroblasts

We have conducted single-channel patch-clamp experiments in skin fibroblasts maintained in culture. Two different cell lines, a mouse 3T3-L1 cell line and a human B17 cell line, were selected for these pilot studies. 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 (> 1Gomega) 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-attached patches, the prevalent type of channel in symmetrical K+ solutions had a conductance of 187 pS. This channel was activated by strong depolarization, and there was usually more than one active channel in the patch. It was blocked by extracellular tetraethylammonium (20 mM), and persisted when external Cl- was replaced by aspartate. In excised inside-out patches bathed in symmetrical K+, this channel was activated by an increase in Ca+ applied to the intracellular face. A large conductance 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 these recording conditions (2 mM Ca(2+)i). We conclude that the channel we have identified in these cells belongs to the maxi-K+ channel class.