The role of N‐, Q‐ and R‐type Ca2+ channels in feedback inhibition of ACh release from rat basal forebrain neurones

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Abstract
1 The Ca2+ channel subtypes controlling ACh release from basal forebrain neurones and the ionic basis underlying muscarinic receptor-mediated autoinhibition were studied using skeletal myoballs to detect ACh release from individual rat basal forebrain neurones in culture. 2 Somatic Ca2+ currents evoked using a simulated action potential waveform revealed that Ca2+ entry was primarily through N-, Q- and to a lesser extent R-, T- and L-type Ca2+ channels. 3 Muscarine (10 μm) inhibited N- and Q- but not R-, T- or L-type somatic Ca2+ channels. Agonist inhibition was totally blocked by pre-treatment with pertussis toxin (500 ng ml−1). 4 ACh release from discrete sites along basal forebrain neurites (1.2 mm extracellular Ca2+) could be largely abolished by blocking Ca2+ entry through either N-type or Q-type Ca2+ channels. Inhibition of Ca2+ entry through L- or T-type channels had no effect upon release. Following inhibition of either N- or Q-type Ca2+ channels, release could be restored to near control levels by raising [Ca2+]o. After selectively blocking N-, Q-, L- and T-type channels, low levels of release could still be evoked as a result of Ca2+ entry through R-type Ca2+ channels. 5 Muscarinic receptor activation reversibly inhibited ACh release due to Ca2+ entry through N-, Q- and R-type Ca2+ channels. In contrast, inhibition of inwardly rectifying K+ channels using Ba2+ (3–10 μm) or substance P (0.03–0.1 μm), or block of SK or BK Ca2+-activated K+ channels with apamin (100 nm) or charbydotoxin (100 nm) respectively, had no effect upon either ACh release or its modulation by muscarinic agonists. 6 These results show that ACh release from individual release sites on basal forebrain neurones is controlled by multiple Ca2+ channel subtypes with overlapping Ca2+ microdomains and that autoinhibition of release results from M2 muscarinic receptor-mediated inhibition of these presynaptic Ca2+ channels rather than as a consequence of K+ channel activation.