The interaction between the I-II loop and the III-IV loop of Cav2.1 contributes to voltage-dependent inactivation in a beta -dependent manner.
We have investigated the molecular mechanisms whereby the I-II loop controls voltage-dependent inactivation in P/Q calcium channels. We demonstrate that the I-II loop is localized in a central position to control calcium channel activity through the interaction with several cytoplasmic sequences; including the III-IV loop. Several experiments reveal the crucial role of the interaction between the I-II loop and the III-IV loop in channel inactivation. First, point mutations of two amino acid residues of the I-II loop of Ca(v)2.1 (Arg-387 or Glu-388) facilitate voltage-dependent inactivation. Second, overexpression of the III-IV loop, or injection of a peptide derived from this loop, produces a similar inactivation behavior than the mutated channels. Third, the III-IV peptide has no effect on channels mutated in the I-II loop. Thus, both point mutations and overexpression of the III-IV loop appear to act similarly on inactivation, by competing off the native interaction between the I-II and the III-IV loops of Ca(v)2.1. As they are known to affect inactivation, we also analyzed the effects of beta subunits on these interactions. In experiments in which the beta(4) subunit is co-expressed, the III-IV peptide is no longer able to regulate channel inactivation. We conclude that (i) the contribution of the I-II loop to inactivation is partly mediated by an interaction with the III-IV loop and (ii) the beta subunits partially control inactivation by modifying this interaction. These data provide novel insights into the mechanisms whereby the beta subunit, the I-II loop, and the III-IV loop altogether can contribute to regulate inactivation in high voltage-activated calcium channels.
Geib
Sandrine
S
INSERM Unité 464, Laboratoire de Neurobiologie des Canaux Ioniques, Faculté de Médecine Nord, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France.
Sandoz
Guillaume
G
Cornet
Veronique
V
Mabrouk
Kamel
K
Fund-Saunier
Odile
O
Bichet
Delphine
D
Villaz
Michel
M
Hoshi
Toshinori
T
Sabatier
Jean-Marc
JM
De Waard
Michel
M
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D016827:CD8 Antigens / Q000096:biosynthesis; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry*; D020864:Calcium Channels, N-Type / Q000096:biosynthesis* / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004594:Electrophysiology; D018698:Glutamic Acid / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010452:Peptide Biosynthesis; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors; D014982:Xenopus laevis
D001120:Arginine; D016827:CD8 Antigens; D002118:Calcium; D015220:Calcium Channels; D020864:Calcium Channels, N-Type; D018698:Glutamic Acid; D005982:Glutathione Transferase; D011993:Recombinant Fusion Proteins