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J. Biol. Chem., Vol. 283, Issue 30, 21036-21044, July 25, 2008

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M₂ Muscarinic Receptors Induce Airway Smooth Muscle Activation via a Dual, Gβ-mediated Inhibition of Large Conductance Ca²⁺-activated K⁺ Channel Activity^*

Xiao-Bo Zhou, Iris Wulfsen, Susanne Lutz, Emine Utku, Ulrike Sausbier^¶, Peter Ruth^¶, Thomas Wieland¹, and Michael Korth¹²

From the Institut für Pharmakologie für Pharmazeuten, Universitätsklinikum Hamburg-Eppendorf, 20246 Hamburg, Germany, the ^¶Pharmakologie und Toxikologie, Pharmazeutisches Institut, Universität Tübingen, 72076 Tübingen, Germany, and the Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Medizinische Fakultät Mannheim, Universität Heidelberg, 68169 Mannheim, Germany

Airway smooth muscle is richly endowed with muscarinic receptors of the M₂ and M₃ subtype. Stimulation of these receptors inhibits large conductance calcium-activated K⁺ (BK) channels, a negative feed back regulator, in a pertussis toxinsensitive manner and thus facilitates contraction. The underlying mechanism, however, is unknown. We therefore studied the activity of bovine trachea BK channels in HEK293 cells expressing the M₂ or M₃ receptor (M₂RorM₃R). In M₂R- but not M₃R-expressing cells, maximal effective concentrations of carbamoylcholine (CCh) inhibited whole cell BK currents by 53%. This M₂R-induced inhibition was abolished by pertussis toxin treatment or overexpression of the Gβ scavenger transducin-. In inside-out patches, direct application of 300 nM purified Gβ decreased channel open probability by 55%. The physical interaction of Gβ with BK channels was confirmed by co-immunoprecipitation. Interestingly, inhibition of phospholipase C as well as protein kinase C activities also reversed the CCh effect but to a smaller (20%) extent. Mouse tracheal cells responded similarly to CCh, purified Gβ and phospholipase C/protein kinase C inhibition as M₂R-expressing HEK293 cells. Our results demonstrate that airway M₂Rs inhibit BK channels by a dual, Gβ-mediated mechanism, a direct membrane-delimited interaction, and the activation of the phospholipase C/protein kinase C pathway.

Received for publication, January 17, 2008 , and in revised form, April 24, 2008.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Institut für Pharmakologie für Pharmazeuten, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany. Tel.: 49-40-428032884; Fax: 49-40-428035761; E-mail: korth{at}uke.uni-hamburg.de.

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