The C-terminal residues in the alpha-interacting domain (AID) helix anchor CaV beta subunit interaction and modulation of CaV2.3 channels.
The alpha-interacting domain (AID) in the I-II linker of high voltage-activated (HVA) Ca(2+) channel alpha1 subunits binds with high affinity to Ca(V)beta auxiliary subunits. The recently solved crystal structures of the AID-Ca(V)beta complex in Ca(V)1.1/1.2 have revealed that this interaction occurs through a set of six mostly invariant residues Glu/Asp(6), Leu(7), Gly(9), Tyr(10), Trp(13), and Ile(14) (where the superscript refers to the position of the residue starting with the QQ signature doublet) distributed among three alpha-helical turns in the proximal section of the I-II linker. We show herein that alanine mutations of N-terminal AID residues Gln(1), Gln(2), Ile(3), Glu(4), Glu(6), Leu(7), and Gly(9) in Ca(V)2.3 did not abolish [(35)S]Ca(V)beta 1b or [(35)S]Ca(V)beta 3 subunit overlay binding to fusion proteins nor did they prevent the typical modulation of whole cell currents by Ca(V)beta 3. Mutations of the invariant Tyr(10) with either hydrophobic (Ala), aromatic (Phe), or positively charged (Arg, Lys) residues yielded Ca(V)beta 3-responsive whole cell currents, whereas mutations with negatively charged residues (Asp, Glu) disrupted Ca(V)beta 3 binding and modulation. In contrast, modulation and binding by Ca(V)beta 3 was significantly weakened in I14A (neutral and hydrophobic) and I14S (neutral and polar) mutants and eradicated in negatively charged I14D and I14E or positively charged I14R and I14K mutants. Ca(V)beta 3-induced modulation was only preserved with the conserved I14L mutation. Molecular replacement analyses carried out using a three-dimensional homology model of the AID helix from Ca(V)2.3 suggests that a high degree of hydrophobicity and a restrained binding pocket could account for the strict structural specificity of the interaction site found at position Ile(14). Altogether these results indicate that the C-terminal residues Trp(13) (1) and Ile(14) anchor Ca(V)beta subunit functional modulation of HVA Ca(2+) channels.
Berrou
Laurent
L
Département de Physiologie, Université de Montréal, Québec H3C 3J7, Canada.
Dodier
Yolaine
Y
Raybaud
Alexandra
A
Tousignant
Audrey
A
Dafi
Omar
O
Pelletier
Joelle N
JN
Parent
Lucie
L
D000409:Alanine; D000818:Animals; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism; D020908:Calcium Channels, R-Type; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D051381:Rats; D013329:Structure-Activity Relationship
C423009:CACNA1E protein, human; C498917:Cacna1e protein, rat; D015220:Calcium Channels; D020908:Calcium Channels, R-Type; D027682:Cation Transport Proteins; D021122:Protein Subunits; D000409:Alanine