Reconstituted slow muscarinic inhibition of neuronal (Ca(v)1.2c) L-type Ca2+ channels.
Ca(2+) influx through L-type channels is critical for numerous physiological functions. Relatively little is known about modulation of neuronal L-type Ca(2+) channels. We studied modulation of neuronal Ca(V)1.2c channels heterologously expressed in HEK293 cells with each of the known muscarinic acetylcholine receptor subtypes. Galphaq/11-coupled M1, M3, and M5 receptors each produced robust inhibition of Ca(V)1.2c, whereas Galphai/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(V)1.2c was blocked by coexpressing RGS2 or RGS3T or by intracellular dialysis with antibodies directed against Galphaq/11. In contrast, inhibition was not reduced by coexpressing betaARK1ct or Galphat. These results indicate that slow inhibition required signaling by Galphaq/11, but not Gbetagamma, subunits. Slow inhibition did not require Ca(2+) transients or Ca(2+) influx through Ca(V)1.2c 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 Ca(2+) chelation. The cardiac splice-variant of Ca(V)1.2 (Ca(V)1.2a) and a splice-variant of the neuronal/neuroendocrine Ca(V)1.3 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.
Bannister
Roger A
RA
Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT 84322, USA.
Melliti
Karim
K
Adams
Brett A
BA
D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000187:drug effects* / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007668:Kidney / Q000187:drug effects / Q000196:embryology / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018727:Muscarinic Antagonists / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000502:physiology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D012680:Sensitivity and Specificity
D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D018727:Muscarinic Antagonists; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D004533:Egtazic Acid; D002217:Carbachol; D037342:Pertussis Toxin; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium