Biophys J, December 2002, p. 3256-3267, Vol. 83, No. 6

Reconstituted Slow Muscarinic Inhibition of Neuronal (Ca_V1.2c) L-Type Ca²⁺ Channels

Roger A. Bannister,^* Karim Melliti, and Brett A. Adams^*

 ^*Department of Biology, Utah State University, Logan, Utah 84322 USA; and  Merck, Sharp, and Dohme Research Laboratories, The Neuroscience Research Center, Terlings Park, Harlow, Essex CM20 2QR, United Kingdom

Ca²⁺ influx through L-type channels is critical for numerous physiological functions. Relatively little is known about modulation of neuronal L-type Ca²⁺ channels. We studied modulation of neuronal Ca_V1.2c channels heterologously expressed in HEK293 cells with each of the known muscarinic acetylcholine receptor subtypes. Gq/11-coupled M1, M3, and M5 receptors each produced robust inhibition of Ca_V1.2c, whereas Gi/o-coupled M2 and M4 receptors were ineffective. Channel inhibition through M1 receptors was studied in detail and was found to be kinetically slow, voltage-independent, and pertussis toxin-insensitive. Slow inhibition of Ca_V1.2c was blocked by coexpressing RGS2 or RGS3T or by intracellular dialysis with antibodies directed against Gq/11. In contrast, inhibition was not reduced by coexpressing ARK1ct or Gt. These results indicate that slow inhibition required signaling by Gq/11, but not G, subunits. Slow inhibition did not require Ca²⁺ transients or Ca²⁺ influx through Ca_V1.2c channels. Additionally, slow inhibition was insensitive to pharmacological inhibitors of phospholipases, protein kinases, and protein phosphatases. Intracellular BAPTA prevented slow inhibition via a mechanism other than Ca²⁺ chelation. The cardiac splice-variant of Ca_V1.2 (Ca_V1.2a) and a splice-variant of the neuronal/neuroendocrine Ca_V1.3 channel also appeared to undergo slow muscarinic inhibition. Thus, slow muscarinic inhibition may be a general characteristic of L-type channels having widespread physiological significance.

Biophys J, December 2002, p. 3256-3267, Vol. 83, No. 6
© 2002 by the Biophysical Society   0006-3495/02/12/3256/12  $2.00

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