4 The suppression of IM by muscarine was similar in cultured cells derived from adult and foetal tissue to that seen in the intact ganglia. 5 IM-suppression by muscarine was inhibited by pirenzepine (Pz) and AF-DX 116 with mean pKB values of 7.53 + 0.13 (n = 3) and 6.02 + 0.13 (n = 4) respectively. 6 The suppression of IM by muscarinic agonists was not affected by gallamine (10-30 pM). 4-Diphenylacetoxy-N-methylpiperidine methiodide inhibited the response at 300 nm. 7 Pirenzepine inhibited the contractions of the guinea-pig isolated ileum produced by muscarine with a mean pKB of 6.37 + 0.03 (n = 8). 8 These results suggest that the receptors mediating suppression of the M-current accord with those designated pharmacologically as M1 and that these receptors reach maturity at a very early stage in the development of the rat s.c.g.

SUMMARY1. The properties of single Ca2+-activated K+ channels in cultured rat superior cervical ganglionic neurones were studied in cell-attached and excised patches using the patch-clamp technique.2. In cell-attached patches using an external K+ concentration ([K+]0) of 150 mM, approximately equal to the internal [K+], the channel slope conductance was -200 pS and independent of membrane voltage between -50 and + 50 mV. Using [K+]. of 4-7 mm (providing a near physiological K+ gradient), the I-V relationship was non-linear with a slope conductance of -120 pS at 0 mV.3. The channel was selective for K+ over Cs+ and Na+ which were impermeant from either side of the membrane. Both Na+ and Cs+ also blocked the movement of K+ through the channel. Cs+ was active on either side of the membrane, whereas Na+ apparently blocked the channel only when applied to the cytoplasmic side.4

SUMMARY1. Dissociated adult or fetal rat superior cervical ganglion cells were voltageclamped through a single patch pipette. The voltage-dependent K+ current, IM (Mcurrent), was maintained by including MgATP in the pipette solution and by buffering the solution pH to 6 7.2. Bath-applied muscarine (0 4 /IM) produced a reversible inhibition of IM.3. Addition of Gpp(NH)p (200 /tM) or GTP-y-S (500 /tM) to the pipette solution induced a slowly developing inhibition ofIM and prevented recovery from subsequent muscarine-induced inhibition.4. Addition of GDP-,/-S (500 uM) to the pipette solution reduced the amount of IM inhibition produced by 04 ,aM-muscarine by 42 % and reduced the associated inward shift of the holding current by 56 %.5. Cells responded normally to muscarine after pre-treatment for 4-27 h with 500 ng ml-1 pertussis toxin (PTx).6. IM was not diminished by extracellular addition of 1 mM-dibutyryl cyclic AMP, 8-bromo-cyclic AMP or dibutyryl cyclic GMP, or of 10 ,uM-forskolin.7. IM was not reduced by inclusion of Li' (2 mM) or inositol 1,4,5-trisphosphate (IP3, 100 /iM) in the patch pipette, nor by ionophoretic injection of 1P3 from an inserted micropipette.8. Addition of 4-,l-phorbol 12,13-dibutyrate (PDBu, 0 5-2 ,UM) to the extracellular medium partly inhibited IM and reduced an additional component of resting membrane current. This effect was not replicated by 4-a-phorbol 12,13-didecanoate. 9. It is concluded that the inhibition of IM by muscarine is mediated through activation of a PTx-insensitive GTP-binding protein. The effect of muscarine appears not to be mediated by cyclic nucleotides or 1P3 but may possibly involve the generation of diacylglycerols and activation of protein kinase C.

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