Inositol trisphosphate mediates cloned muscarinic receptor-activated conductances in transfected mouse fibroblast A9 L cells.
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 reduced the ACh-induced Ca2+ response. 7. The ACh-induced currents recorded at -50 mV were unaffected by removal of extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)
Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
S V
SV
Jones
J L
JL
Barker
M B
MB
Goodman
M R
MR
Brann
D000109:Acetylcholine / Q000494:pharmacology; D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002712:Chlorides / Q000502:physiology; D002999:Clone Cells / Q000502:physiology; D005347:Fibroblasts / Q000502:physiology; D005456:Fluorescent Dyes; D016257:Fura-2; D006160:Guanosine Triphosphate / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D007473:Ion Channels / Q000187:drug effects; D051379:Mice; D011188:Potassium / Q000502:physiology; D051381:Rats; D011976:Receptors, Muscarinic / Q000502:physiology*; D014162:Transfection
D001572:Benzofurans; D002712:Chlorides; D005456:Fluorescent Dyes; D007473:Ion Channels; D011976:Receptors, Muscarinic; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D000109:Acetylcholine; D011188:Potassium; D002118:Calcium; D016257:Fura-2