Neuropeptide modulation of single calcium and potassium channels detected with a new patch clamp configuration.
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 A pipette containing the pore-forming agent nystatin is sealed onto a cell and withdrawn to form an enclosed vesicle.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 dihydropyridine-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.
Levitan
E S
ES
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Kramer
R H
RH
D002118:Calcium / Q000502:physiology; D015220:Calcium Channels / Q000502:physiology*; D002460:Cell Line; D004594:Electrophysiology / Q000379:methods*; D066298:In Vitro Techniques; D008564:Membrane Potentials; D009761:Nystatin / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000502:physiology; D010902:Pituitary Gland / Q000166:cytology; D015221:Potassium Channels / Q000502:physiology*; D015398:Signal Transduction; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*
D015220:Calcium Channels; D010716:Phosphatidylinositols; D015221:Potassium Channels; D009761:Nystatin; D013973:Thyrotropin-Releasing Hormone; D002118:Calcium