Direct visualization of interaction between calmodulin and connexin45

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.

Ca2+ Cycling Impairment in Heart Failure Is Exacerbated by Fibrosis: Insights Gained From Mechanistic Simulations

“…Gap junctional coupling between a myocyte and a fibroblast has been modeled by a simple conductance as in previous simulation studies of fibroblast-myocyte coupling. However, recent findings suggesting the interaction of connexin45 with CaM ( Zou et al, 2017 ) highlight that some ions such as Ca 2+ can modulate the coupling current. If the modulation of the intercellular coupling by Ca 2+ ions was taken into account, larger alterations in Ca 2+ dynamics mechanisms could be observed.…”

Calcium-Calmodulin Gating of Connexin43 Gap Junctions in the Absence of the pH Gating Domain

“…The existence of Cx CaMBDs has been demonstrated by in vitro CaM binding assays with sequence-specific Cx mimetic peptides comprising the entire15-26 amino acid CaMBDs of Cx32, Cx43, sheep Cx44 (Cx46), Cx45, and Cx50 (17)(18)(19)(20)(21). In past studies, these Cx CaMBD mimetic peptides were used as inhibitory peptides to validate the functional role of the Cxspecific sequence in the Ca 2+ /CaM gating of the parent connexin (20,22).…”

“…The calcium gating hypothesis evolved to the action of calmodulin (CaM) based on the inhibition of gap junction uncoupling by calmodulin (CaM) inhibitors and evidence that CaM binds to Cx32 and lens gap junctions (15)(16)(17). Since the identification of CaM binding domains (CaMBDs) on the cytoplasmic amino-and carboxyl termini of Cx32, additional connexin CaMBDs have been identified on the CL domain of Cx43, Cx50, Cx46 (sheep Cx44), and Cx45 (18)(19)(20)(21). Previous studies in this laboratory indicated that Ca 2+ /CaM causes a gated closure of Cx43 and Cx50 gap junctions as evidenced by the reduced open probability of gap junction channels during perfusion of coupled cell pairs with the calcium ionophore ionomycin (20,22).…”

Heterocellular Coupling Between Amacrine Cells and Ganglion Cells

“…The primary proteins of gap junctions are drawn from a large family of connexins with four transmembrane spanning domains, cytosolic domains that usually (though not always) provide predominantly homotypic or bihomotypic binding even if the junctions are heteromeric ( Li et al, 2008 ; Rash et al, 2013 ), and intracellular domains that mediate recognition and binding of other gap junction proteins. In general, it is thought that the peak open conductance of a single connexon is principally related to its pore diameter (this is not always true) with complex modulation enabled by a range of mechanisms ( Ek-Vitorin and Burt, 2013 ; Hervé and Derangeon, 2013 ) including connexin phosphorylation ( Pereda et al, 2013 ; O’Brien, 2017 ), methanesulfonate-analog (taurine) binding ( Locke et al, 2011 ), and many different adapter protein interactions (e.g., Zou et al, 2017 ). Light-induced changes in gap junctions are currently understood to modify the open conductance of a connexon through these mechanisms, but will not change the presence or absence of gap junctions at contact sites with coupling partners.…”