The role of distal S6 hydrophobic residues in the voltage-dependent gating of CaV2.3 channels.
The hydrophobic locus VAVIM is conserved in the S6 transmembrane segment of domain IV (IVS6) in Ca(V)1 and Ca(V)2 families. Herein we show that glycine substitution of the VAVIM motif in Ca(V)2.3 produced whole cell currents with inactivation kinetics that were either slower (A1719G approximately V1720G), similar (V1718G), or faster (I1721G approximately M1722G) than the wild-type channel. The fast kinetics of I1721G were observed with a approximately +10 mV shift in its voltage dependence of activation (E(0.5,act)). In contrast, the slow kinetics of A1719G and V1720G were accompanied by a significant shift of approximately -20 mV in their E(0.5,act) indicating that the relative stability of the channel closed state was decreased in these mutants. Glycine scan performed with Val (349) in IS6, Ile(701) in IIS6, and Leu(1420) in IIIS6 at positions predicted to face Val(1720) in IVS6 also produced slow inactivating currents with hyperpolarizing shifts in the activation and inactivation potentials, again pointing out a decrease in the stability of the channel closed state. Mutations to other hydrophobic residues at these positions nearly restored the channel gating. Altogether these data indicate that residues at positions equivalent to 1720 exert a critical control upon the relative stability of the channel closed and open states and more specifically, that hydrophobic residues at these positions promote the channel closed state. We discuss a three-dimensional homology model of Ca(V)2.3 based upon Kv1.2 where hydrophobic residues at positions facing Val(1720) in IS6, IIS6, and IIIS6 play a critical role in stabilizing the closed state in Ca(V)2.3.
Département de Physiologie and the Membrane Protein Research Group, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
Alexandra
A
Raybaud
Ebru-Eylem
EE
Baspinar
François
F
Dionne
Yolaine
Y
Dodier
Rémy
R
Sauvé
Lucie
L
Parent
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D020908:Calcium Channels, R-Type / Q000737:chemistry*; D027682:Cation Transport Proteins / Q000737:chemistry*; D004252:DNA Mutational Analysis; D006801:Humans; D007700:Kinetics; D051663:Kv1.2 Potassium Channel / Q000737:chemistry; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014981:Xenopus
C423009:CACNA1E protein, human; D020908:Calcium Channels, R-Type; D027682:Cation Transport Proteins; D051663:Kv1.2 Potassium Channel; D011994:Recombinant Proteins