Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 280, Issue 1, Pages 494-505Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M410859200
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The alpha-interacting domain (AID) in the I-II linker of high voltage-activated (HVA) Ca2+ 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 [S-35]Ca(v)beta1b or [S-35]Ca(v)beta3 subunit overlay binding to fusion proteins nor did they prevent the typical modulation of whole cell currents by Ca(v)beta3. Mutations of the invariant Tyr(10) with either hydrophobic (Ala), aromatic (Phe), or positively charged (Arg, Lys) residues yielded Ca(v)beta3-responsive whole cell currents, whereas mutations with negatively charged residues (Asp, Glu) disrupted Ca(v)beta3 binding and modulation. In contrast, modulation and binding by Ca(v)beta3 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)beta3-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 Ile14. Altogether these results indicate that the C-terminal residues Trp13 (1) and Ile(14) anchor Ca(v)beta subunit functional modulation of HVA Ca2+ channels.
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