Proteolytic cleavage of the voltage-gated Ca2+ channel alpha2delta subunit: structural and functional features.
By mediating depolarization-induced Ca(2+) influx, high-voltage-activated Ca(2+) channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (alpha(1)) and three auxiliary (alpha(2)delta, beta and gamma) subunits. The alpha(2)delta subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, alpha(2)delta must undergo important post-translational modifications, including a proteolytic cleavage that separates the extracellular alpha(2) from its transmembrane delta domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature alpha(2)delta, almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of alpha(2)delta was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide-reducing agent. Interestingly, these mutations significantly decreased whole-cell patch-clamp currents without affecting the voltage dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane.
Andrade
Arturo
A
Department of Physiology, Biophysics and Neuroscience, Cinvestav, Mexico City, Mexico.
Sandoval
Alejandro
A
Oviedo
Norma
N
De  Waard
Michel
M
Elias
David
D
Felix
Ricardo
R
D020816:Amino Acid Motifs; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D004558:Electric Stimulation / Q000379:methods; D015870:Gene Expression; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology / Q000528:radiation effects; D016296:Mutagenesis / Q000502:physiology; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D014162:Transfection
D015220:Calcium Channels; C494035:voltage gated calcium channel subunit Ca alpha2delta-1, human