Jones, Susan; (1995) G proteins transducing receptor-mediated inhibition of the M-type K+ current in rat cultured sympathetic neurones. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In rat sympathetic neurones, the M-type K+ current (IK(M)) can be inhibited via receptors that couple to G proteins. This thesis describes the identification of these G proteins. The perforated patch voltage-clamp method was used to record IK(M) from rat cultured superior cervical ganglion (SCG) neurones. IK(M) inhibition by the muscarinic receptor agonist, oxotremorine-M (oxo-M; EC50 204 nM), and the peptide hormone, bradykinin (BK; EC50 0.95 nM), was recorded. To identify G proteins transducing IK(M) inhibition, anti-Gα protein (C-terminal sequence) antibodies were used. Microinjection of anti-Gαq/11 antibody caused a significant but variable reduction of IK(M) inhibition by 300 nM oxo-M; microinjection of anti-Gαo antibody had no effect on the response to oxo-M. The effect of the anti-Gαq/11 antibody was surmountable on increasing the concentration of oxo-M, suggesting a spare G protein capacity. To ascertain whether Gαq, Gα11, or both transduce muscarinic IK(M) inhibition, antisense DNA sequences were used. Although initial results suggested a significant role for Gα11, these experiments were not reproducible. BK potently inhibited IK(M) in SCG neurones, and this effect of BK was also reduced by anti-Gαq/11 (but not anti-Gαo) antibody, suggesting that BK receptors and muscarinic receptors use a common or closely related mechanism to inhibit IK(M)- However, there were differences in the response to the two agonists: the response to BK was slower in onset and offset, generally showed less recovery than the response to oxo-M, and the effect of high concentrations of BK, but not oxo-M, was susceptible to tachyphylaxis. Additionally, the effect of anti-Gαq/11 antibody on IK(M) inhibition by BK was not as variable as the effect on the response to oxo-M. It is concluded that muscarinic and BK receptors can couple to the same or highly homologous G proteins (Gαq/11) to inhibit IK(M)- The different characteristics of the responses to the two agonists are discussed.

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