title: Intramembrane charge movements and excitation- contraction coupling expressed by two-domain fragments of the Ca2+ channel.
abstract: To investigate the molecular basis of the voltage sensor that triggers excitation-contraction (EC) coupling, the four-domain pore subunit of the dihydropyridine receptor (DHPR) was cut in the cytoplasmic linker between domains II and III. cDNAs for the I-II domain (alpha1S 1-670) and the III-IV domain (alpha1S 701-1873) were expressed in dysgenic alpha1S-null myotubes. Coexpression of the two fragments resulted in complete recovery of DHPR intramembrane charge movement and voltage-evoked Ca(2+) transients. When fragments were expressed separately, EC coupling was not recovered. However, charge movement was detected in the I-II domain expressed alone. Compared with I-II and III-IV together, the charge movement in the I-II domain accounted for about half of the total charge (Q(max) = 3 +/- 0.23 vs. 5.4 +/- 0.76 fC/pF, respectively), and the half-activation potential for charge movement was significantly more negative (V(1/2) = 0.2 +/- 3.5 vs. 22 +/- 3.4 mV, respectively). Thus, interactions between the four internal domains of the pore subunit in the assembled DHPR profoundly affect the voltage dependence of intramembrane charge movement. We also tested a two-domain I-II construct of the neuronal alpha1A Ca(2+) channel. The neuronal I-II domain recovered charge movements like those of the skeletal I-II domain but could not assist the skeletal III-IV domain in the recovery of EC coupling. The results demonstrate that a functional voltage sensor capable of triggering EC coupling in skeletal myotubes can be recovered by the expression of complementary fragments of the DHPR pore subunit. Furthermore, the intrinsic voltage-sensing properties of the alpha1A I-II domain suggest that this hemi-Ca(2+) channel could be relevant to neuronal function.
authors.affiliation: Department of Physiology, University of Wisconsin School of Medicine, Madison, WI 53706, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Ahern
authors.forename: J
authors.initials: J
authors.lastname: Arikkath
authors.forename: P
authors.initials: P
authors.lastname: Vallejo
authors.forename: C A
authors.initials: CA
authors.lastname: Gurnett
authors.forename: P A
authors.initials: PA
authors.lastname: Powers
authors.forename: K P
authors.initials: KP
authors.lastname: Campbell
authors.forename: R
authors.initials: R
authors.lastname: Coronado
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000502:physiology; D020746:Calcium Channels, L-Type / Q000032:analysis / Q000737:chemistry*; D051379:Mice; D009119:Muscle Contraction; D010446:Peptide Fragments / Q000737:chemistry* / Q000502:physiology; D014162:Transfection
chemical_list: D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D010446:Peptide Fragments; D002118:Calcium