Molecular Basis of Regulating High Voltage-Activated Calcium Channels by S-Nitrosylation.
Nitric oxide (NO) is involved in a variety of physiological processes, such as vasoregulation and neurotransmission, and has a complex role in the regulation of pain transduction and synaptic transmission. We have shown previously that NO inhibits high voltage-activated Ca(2+) channels in primary sensory neurons and excitatory synaptic transmission in the spinal dorsal horn. However, the molecular mechanism involved in this inhibitory action remains unclear. In this study, we investigated the role of S-nitrosylation in the NO regulation of high voltage-activated Ca(2+) channels. The NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) rapidly reduced N-type currents when Cav2.2 was coexpressed with the Cavβ1 or Cavβ3 subunits in HEK293 cells. In contrast, SNAP only slightly inhibited P/Q-type and L-type currents reconstituted with various Cavβ subunits. SNAP caused a depolarizing shift in voltage-dependent N-type channel activation, but it had no effect on Cav2.2 protein levels on the membrane surface. The inhibitory effect of SNAP on N-type currents was blocked by the sulfhydryl-specific modifying reagent methanethiosulfonate ethylammonium. Furthermore, the consensus motifs of S-nitrosylation were much more abundant in Cav2.2 than in Cav1.2 and Cav2.1. Site-directed mutagenesis studies showed that Cys-805, Cys-930, and Cys-1045 in the II-III intracellular loop, Cys-1835 and Cys-2145 in the C terminus of Cav2.2, and Cys-346 in the Cavβ3 subunit were nitrosylation sites mediating NO sensitivity of N-type channels. Our findings demonstrate that the consensus motifs of S-nitrosylation in cytoplasmically accessible sites are critically involved in post-translational regulation of N-type Ca(2+) channels by NO. S-Nitrosylation mediates the feedback regulation of N-type channels by NO.
From the Department of Anesthesiology and Perioperative Medicine, Center for Neuroscience and Pain Research, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030.
Meng-Hua
MH
Zhou
From the Department of Anesthesiology and Perioperative Medicine, Center for Neuroscience and Pain Research, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030.
Alexis
A
Bavencoffe
From the Department of Anesthesiology and Perioperative Medicine, Center for Neuroscience and Pain Research, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030 huilinpan@mdanderson.org.
Hui-Lin
HL
Pan
D020816:Amino Acid Motifs; D000818:Animals; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology
D020864:Calcium Channels, N-Type; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; C500545:CACNA1B protein, human; C503279:Cacna1b protein, rat
calcium channel; ion channel; nitric oxide; nitrosylation; pain; post-transcriptional regulation