Dataset Information

Ca²⁺ Dynamics of Gap Junction Coupled and Uncoupled Deiters' Cells in the Organ of Corti in Hearing BALB/c Mice.

ABSTRACT: ATP, as a paracrine signalling molecule, induces intracellular Ca²⁺ elevation via the activation of purinergic receptors on the surface of glia-like cochlear supporting cells. These cells, including the Deiters' cells (DCs), are also coupled by gap junctions that allow the propagation of intercellular Ca²⁺ waves via diffusion of Ca²⁺ mobilising second messenger IP₃ between neighbouring cells. We have compared the ATP-evoked Ca²⁺ transients and the effect of two different gap junction (GJ) blockers (octanol and carbenoxolone, CBX) on the Ca²⁺ transients in DCs located in the apical and middle turns of the hemicochlea preparation of BALB/c mice (P14-19). Octanol had no effect on Ca²⁺ signalling, while CBX inhibited the ATP response, more prominently in the middle turn. Based on astrocyte models and using our experimental results, we successfully simulated the Ca²⁺ dynamics in DCs in different cochlear regions. The mathematical model reliably described the Ca²⁺ transients in the DCs and suggested that the tonotopical differences could originate from differences in purinoceptor and Ca²⁺ pump expressions and in IP₃-Ca²⁺ release mechanisms. The cochlear turn-dependent effect of CBX might be the result of the differing connexin isoform composition of GJs along the tonotopic axis. The contribution of IP₃-mediated Ca²⁺ signalling inhibition by CBX cannot be excluded.

SUBMITTER: Moysan L

PROVIDER: S-EPMC10342047 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Ca2+ Dynamics of Gap Junction Coupled and Uncoupled Deiters' Cells in the Organ of Corti in Hearing BALB/c Mice.

Moysan Louise L Fazekas Fruzsina F Fekete Adam A Köles László L Zelles Tibor T Berekméri Eszter E

International journal of molecular sciences 20230704 13

ATP, as a paracrine signalling molecule, induces intracellular Ca2+ elevation via the activation of purinergic receptors on the surface of glia-like cochlear supporting cells. These cells, including the Deiters' cells (DCs), are also coupled by gap junctions that allow the propagation of intercellular Ca2+ waves via diffusion of Ca2+ mobilising second messenger IP3 between neighbouring cells. We have compared the ATP-evoked Ca2+ transients a ...[more]

PMID: 37446272

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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.

Project description: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.

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Dataset Information

Ca²⁺ Dynamics of Gap Junction Coupled and Uncoupled Deiters' Cells in the Organ of Corti in Hearing BALB/c Mice.

Publications

Ca<sup>2+</sup> Dynamics of Gap Junction Coupled and Uncoupled Deiters' Cells in the Organ of Corti in Hearing BALB/c Mice.

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