Tran, O; Kerruth, S; Coates, C; Kaur, H; Peracchia, C; Carter, T; Török, K
(2023)
Ca2+-Dependent and -Independent Calmodulin Binding to the Cytoplasmic Loop of Gap Junction Connexins.
International Journal of Molecular Sciences, 24 (4).
p. 4153.
ISSN 1422-0067
https://doi.org/10.3390/ijms24044153
SGUL Authors: Carter, Thomas David Torok, Katalin
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Abstract
Ca2+/calmodulin (Ca2+/CaM) interaction with connexins (Cx) is well-established; however, the mechanistic basis of regulation of gap junction function by Ca2+/CaM is not fully understood. Ca2+/CaM is predicted to bind to a domain in the C-terminal portion of the intracellular loop (CL2) in the vast majority of Cx isoforms and for a number of Cx-s this prediction has proved correct. In this study, we investigate and characterise both Ca2+/CaM and apo-CaM binding to selected representatives of each of the α, β and γ connexin family to develop a better mechanistic understanding of CaM effects on gap junction function. The affinity and kinetics Ca2+/CaM and apo-CaM interactions of CL2 peptides of β-Cx32, γ-Cx35, α-Cx43, α-Cx45 and α-Cx57 were investigated. All five Cx CL2 peptides were found to have high affinity for Ca2+/CaM with dissociation constants (Kd(+Ca)) from 20 to 150 nM. The limiting rate of binding and the rates of dissociation covered a broad range. In addition, we obtained evidence for high affinity Ca2+-independent interaction of all five peptides with CaM, consistent with CaM remaining anchored to gap junctions in resting cells. However, for the α-Cx45 and α-Cx57 CL2 peptides, Ca2+-dependent association at resting [Ca2+] of 50–100 nM is indicated in these complexes as one of the CaM Ca2+ binding sites displays high affinity with Kd of 70 and 30 nM for Ca2+, respectively. Furthermore, complex conformational changes were observed in peptide-apo-CaM complexes with the structure of CaM compacted or stretched by the peptide in a concentration dependent manner suggesting that the CL2 domain may undergo helix-to-coil transition and/or forms bundles, which may be relevant in the hexameric gap junction. We demonstrate inhibition of gap junction permeability by Ca2+/CaM in a dose dependent manner, further cementing Ca2+/CaM as a regulator of gap junction function. The motion of a stretched CaM–CL2 complex compacting upon Ca2+ binding may bring about the Ca2+/CaM block of the gap junction pore by a push and pull action on the CL2 C-terminal hydrophobic residues of transmembrane domain 3 (TM3) in and out of the membrane.
Item Type: | Article | ||||||
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Additional Information: | © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). | ||||||
Keywords: | 0399 Other Chemical Sciences, 0604 Genetics, 0699 Other Biological Sciences, Chemical Physics | ||||||
SGUL Research Institute / Research Centre: | Academic Structure > Molecular and Clinical Sciences Research Institute (MCS) | ||||||
Journal or Publication Title: | International Journal of Molecular Sciences | ||||||
ISSN: | 1422-0067 | ||||||
Language: | en | ||||||
Dates: |
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Publisher License: | Creative Commons: Attribution 4.0 | ||||||
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URI: | https://openaccess.sgul.ac.uk/id/eprint/115199 | ||||||
Publisher's version: | https://doi.org/10.3390/ijms24044153 |
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