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

Abstract

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)