Neuropeptide modulation of single calcium and potassium channels detected with a new patch clamp configuration

Abstract

CALCIUM channel activity is crucial for secretion and synaptic transmission, but it has been difficult to study Ca²⁺ channel modulation because survival and regulation of some of these channels require cytoplasmic constituents that are lost with the formation of cell-free patches. Here we report a new patch clamp configuration in which activity and regulation of channels are maintained after removal from cells. A pipette containing the pore-forming agent nystatin is sealed onto a cell and withdrawn to form an enclosed vesicle. The resulting perforated vesicle, formed from pituitary tumour cells, contains Ca²⁺ and K⁺ channels. Ca²⁺-activated K⁺ channels in the vesicle are activated by cyclic AMP analogues, and by a neuropeptide (thyrotropin-releasing hormone) that stimulates phosphatidylinositol turnover and inositol trisphosphate-gated Ca²⁺ release from intracellular organelles. Thus, the perforated vesicle retains signal transduction systems necessary for ion channel modulation. Functional dihy-dropyridine-sensitive Ca²⁺ channels (L-type) are maintained in the vesicle, and their gating is inhibited by thyrotropin-releasing hormone. Hence, this new patch clamp configuration has allowed a direct detection of the single-channel basis of transmitter-induced inhibition of L-type Ca²⁺ channels. The modulation of Ca²⁺-channel gating may be an important mechanism for regulating hormone secretion from pituitary cells.