Direct visualization of interaction between calmodulin and connexin45
- 27 November 2017
- journal article
- research article
- Published by Portland Press Ltd. in Biochemical Journal
- Vol. 474 (24), 4035-4051
- https://doi.org/10.1042/bcj20170426
Abstract
Calmodulin (CaM) is an intracellular Ca2+ transducer involved in numerous activities in a broad Ca2+ signaling network. Previous studies have suggested that the Ca2+/CaM complex may participate in gap junction regulation via interaction with putative CaM-binding motifs in connexins; however, evidence of direct interactions between CaM and connexins has remained elusive to date due to challenges related to the study of membrane proteins. Here, we report the first direct interaction of CaM with Cx45 (connexin45) of γ-family in living cells under physiological conditions by monitoring bioluminescence resonance energy transfer. The interaction between CaM and Cx45 in cells is strongly dependent on intracellular Ca2+ concentration and can be blocked by the CaM inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7). We further reveal a CaM-binding site at the cytosolic loop (residues 164–186) of Cx45 using a peptide model. The strong binding (Kd ∼ 5 nM) observed between CaM and Cx45 peptide, monitored by fluorescence-labeled CaM, is found to be Ca2+-dependent. Furthermore, high-resolution nuclear magnetic resonance spectroscopy reveals that CaM and Cx45 peptide binding leads to global chemical shift changes of 15N-labeled CaM, but does not alter the size of the structure. Observations involving both N- and C-domains of CaM to interact with the Cx45 peptide differ from the embraced interaction with Cx50 from another connexin family. Such interaction further increases Ca2+ sensitivity of CaM, especially at the N-terminal domain. Results of the present study suggest that both helicity and the interaction mode of the cytosolic loop are likely to contribute to CaM's modulation of connexins.This publication has 102 references indexed in Scilit:
- Functional Characterization of Alternative Splicing in the C Terminus of L-type CaV1.3 ChannelsOnline Journal of Public Health Informatics, 2011
- Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of CaV1.2Biophysical Chemistry, 2011
- Structural and Energetic Determinants of Apo Calmodulin Binding to the IQ Motif of the NaV1.2 Voltage-Dependent Sodium ChannelStructure, 2011
- Molecular interaction and functional regulation of connexin50 gap junctions by calmodulinBiochemical Journal, 2011
- Determinants in CaV1 Channels That Regulate the Ca2+ Sensitivity of Bound CalmodulinOnline Journal of Public Health Informatics, 2009
- Calmodulin Mediates the Ca2+-Dependent Regulation of Cx44 Gap JunctionsBiophysical Journal, 2009
- The neuronal connexin36 interacts with and is phosphorylated by CaMKII in a way similar to CaMKII interaction with glutamate receptorsProceedings of the National Academy of Sciences of the United States of America, 2008
- Calmodulin, Conformational States, and Calcium Signaling. A Single-Molecule PerspectiveBiochemistry, 2006
- Biphasic Ca2+-Dependent Switching in a Calmodulin−IQ Domain ComplexBiochemistry, 2006
- NMRPipe: A multidimensional spectral processing system based on UNIX pipesJournal of Biomolecular NMR, 1995