CA-DEPENDENT K-CHANNELS IN SMOOTH-MUSCLE CELLS PERMEABILIZED BY BETA-ESCIN RECORDED USING THE CELL-ATTACHED PATCH-CLAMP TECHNIQUE

Using the cell-attached patch-clamp technique, the activity of single, Ca-dependent K channels was recorded in single smooth muscle cells permeabilized by beta-escin. The conductance and the relationship between the open probability of the channels and pCa recorded in permeabilized cells were very similar to those obtained in excised inside-out patches. At pCa 7, application of 30-mu-M acetylcholine (ACh) or 0.1-mu-M substance P (SP) together with 1 mM guanosine 5'-trisphosphate to permeabilized cells elicited transient bursts of channel openings similar to those which occur in intact cells. Transient activation was also observed when 2-30-mu-M inositol trisphosphate (IP3) was applied to permeabilized cells. This single channel activity was inhibited by pre-treatment with low-molecular-weight heparin at 50-100-mu/ml. Channel activity at pCa 7.0 was greatly enhanced by 200-mu-M cyclic adenosine monophosphate. These results provide direct evidence that single Ca-dependent K channel activity is regulated by the transmitters ACh and SP, as well as a second messenger, IP3, via the release of intracellular Ca from intracellular sites which are blocked by heparin. This novel approach is valuable in elucidating second messenger mechanisms involved in the regulation of single channel activity by transmitters and autacoids, since permeabilization by beta-escin preserves the entire system of receptor-operated signal transduction and allows intracellular application of second messengers at fixed concentrations.