Calmodulin association with connexin32-derived peptides suggests trans-domain interaction in chemical gating of gap junction channels.
Calmodulin plays a key role in the chemical gating of gap junction channels. Two calmodulin-binding regions have previously been identified in connexin32 gap junction protein, one in the N-terminal and another in the C-terminal cytoplasmic tail of the molecule. The aim of this study was to better understand how calmodulin interacts with the connexin32-binding domains. Lobe-specific interactions of calmodulin with connexin32 peptides were studied by stopped flow kinetics, using Ca(2+) binding-deficient mutants. Peptides corresponding to the N-terminal tail (residues 1-22) of connexin32 engaged both the N- and C-terminal lobes (N- and C-lobes) of calmodulin, binding with higher affinity to the C-lobe of calmodulin (Ca(2+) dissociation rate constants k(3,4), 1.7+/-0.5 s(-1)) than to the N-lobe (k(1,2), 10.8+/-1.3 s(-1)). In contrast, peptides representing the C-terminal tail domain (residues 208-227) of connexin32 bound either the C- or the N-lobe but only one calmodulin lobe at a time (k(3,4), 2.6+/-0.1 s(-1) or k(1), 13.8+/-0.5 s(-1) and k(2), 1000 s(-1)). The calmodulin-binding domains of the N- and C-terminal tails of connexin32 were best defined as residues 1-21 and 216-227, respectively. Our data, showing separate functions of the N- and C-lobes of calmodulin in the interactions with connexin32, suggest trans-domain or trans-subunit bridging by calmodulin as a possible mechanism of gap junction gating.
Dodd
Ryan
R
Division of Basic Medical Sciences, St George's, University of London, London, SW17 0RE United Kingdom.
Peracchia
Camillo
C
Stolady
Daniel
D
Török
Katalin
K
D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017630:Connexins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017629:Gap Junctions / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D000097002:Gap Junction beta-1 Protein
D002147:Calmodulin; D017630:Connexins; D000097002:Gap Junction beta-1 Protein