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Ca²⁺-Dependent and -Independent Calmodulin Binding to the Cytoplasmic Loop of Gap Junction Connexins.

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 (K_d(+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 K_d 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.

SUBMITTER: Tran O

PROVIDER: S-EPMC9961272 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Ca2+-Dependent and -Independent Calmodulin Binding to the Cytoplasmic Loop of Gap Junction Connexins.

Tran Oanh O Kerruth Silke S Coates Catherine C Kaur Hansween H Peracchia Camillo C Carter Tom T Török Katalin K

International journal of molecular sciences 20230219 4

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 ...[more]

PMID: 36835569

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Project description:Understanding how cell subpopulations in a tissue impact overall system function is challenging. There is extensive heterogeneity among insulin-secreting β-cells within islets of Langerhans, including their insulin secretory response and gene expression profile, and this heterogeneity can be altered in diabetes. Several studies have identified variations in nutrient sensing between β-cells, including glucokinase (GK) levels, mitochondrial function, or expression of genes important for glucose metabolism. Subpopulations of β-cells with defined electrical properties can disproportionately influence islet-wide free-calcium activity ([Ca2+]) and insulin secretion via gap-junction electrical coupling. However, it is poorly understood how subpopulations of β-cells with altered glucose metabolism may impact islet function. To address this, we utilized a multicellular computational model of the islet in which a population of cells deficient in GK activity and glucose metabolism was imposed on the islet or in which β-cells were heterogeneous in glucose metabolism and GK kinetics were altered. This included simulating GK gene (GCK) mutations that cause monogenic diabetes. We combined these approaches with experimental models in which gck was genetically deleted in a population of cells or GK was pharmacologically inhibited. In each case, we modulated gap-junction electrical coupling. Both the simulated islet and the experimental system required 30-50% of the cells to have near-normal glucose metabolism, fewer than cells with normal KATP conductance. Below this number, the islet lacked any glucose-stimulated [Ca2+] elevations. In the absence of electrical coupling, the change in [Ca2+] was more gradual. As such, electrical coupling allows a large minority of cells with normal glucose metabolism to promote glucose-stimulated [Ca2+]. If insufficient numbers of cells are present, which we predict can be caused by a subset of GCK mutations that cause monogenic diabetes, electrical coupling exacerbates [Ca2+] suppression. This demonstrates precisely how metabolically heterogeneous β-cell populations interact to impact islet function.

Dataset Information

Ca²⁺-Dependent and -Independent Calmodulin Binding to the Cytoplasmic Loop of Gap Junction Connexins.

Publications

Ca<sup>2+</sup>-Dependent and -Independent Calmodulin Binding to the Cytoplasmic Loop of Gap Junction Connexins.

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