Inositol trisphosphate mediates cloned muscarinic receptor-activated conductances in transfected mouse fibroblast A9 L cells

• Published in: The Journal of physiology 1990-02, Vol.421 (1), p.499-519
• Main Authors: Jones, S V, Barker, J L, Goodman, M B, Brann, M R
• Format: Article
• Language: English
• Subjects: Acetylcholine - pharmacology; Action Potentials - drug effects; Animals; Benzofurans; Biological and medical sciences; Calcium - pharmacology; Cell Line; Cell receptors; Cell structures and functions; Chlorides - physiology; Clone Cells - physiology; Fibroblasts - physiology; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Fura-2; Guanosine Triphosphate - pharmacology; Inositol 1,4,5-Trisphosphate - pharmacology; Ion Channels - drug effects; Mice; Molecular and cellular biology; Potassium - physiology; Rats; Receptors, Muscarinic - physiology; Transfection
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.1990.sp017958

1. The mechanism by which cloned m1 and m3 muscarinic receptor subtypes activate Ca2+-dependent channels was investigated with whole-cell and cell-attached patch-clamp recording techniques and with Fura-2 Ca2+ indicator dye measurements in cultured A9 L cells transfected with rat m1 and m3 cDNAs. 2. The Ca2+-dependent K+ and Cl- currents induced by muscarinic receptor stimulation were dependent on GTP. Responses were reduced when GTP was excluded from the intracellular recording solution or when GDP-beta-S was added. Intracellular GTP-gamma-S activated spontaneous fluctuations and permitted only one acetylcholine-(ACh) induced current response. These results implicate GTP-binding proteins (G protein) in the signal transduction pathway. This G protein is probably not pertussis toxin-sensitive as the ACh-induced electrical response was not abolished by pertussis toxin treatment. 3. Cell-attached single-channel recordings revealed activation of ion channels within the patch during application of ACh outside the patch, implying that second messengers might be involved in the ACh-induced response. Two types of K+ channel were activated, a discrete channel of 36 pS and channel activity calculated to be about 5 pS. 4. Application of 8-bromo cyclic AMP or 1-oleoyl-1,2-acetylglycerol (OAG) produced no electrical response and did not affect the ACh-induced responses. Phorbol myristic acetate (PMA) evoked no electrical response, but reduced the ACh-induced responses. 5. Inclusion of inositol 1,4,5-trisphosphate (IP3) in the intracellular pipette solution activated outward currents at -50 mV associated with an increase in conductance. The IP3-induced current response reversed polarity at -65 mV and showed a dependence on K+. Increasing the intracellular free Ca2+ concentration ([Ca2+]i) from 20 nM to 1 microM also induced an outward current response associated with an increase in conductance. Inclusion of inositol 1,3,4,5-tetrakisphosphate (IP4) in the intracellular solution had no effect on the A9 L cells. 6. Fura-2 measurements revealed ACh-induced increases in Cai2+. The Ca2+ responses were abolished by atropine showing that they were muscarinic in nature. Removal of extracellular Ca2+ did not affect the initial ACh-induced increase in Cai2+ but subsequent Cai2+ responses to ACh were depressed, suggesting depletion of Ca2+ intracellular stores. Residual though small responses continued to be elicited by ACh. Barium (5 mM) had little effect and cobalt slightly reduc