Dataset Information

In Vitro Models of GJB2-Related Hearing Loss Recapitulate Ca²⁺ Transients via a Gap Junction Characteristic of Developing Cochlea.

ABSTRACT: Mutation of the Gap Junction Beta 2 gene (GJB2) encoding connexin 26 (CX26) is the most frequent cause of hereditary deafness worldwide and accounts for up to 50% of non-syndromic sensorineural hearing loss cases in some populations. Therefore, cochlear CX26-gap junction plaque (GJP)-forming cells such as cochlear supporting cells are thought to be the most important therapeutic target for the treatment of hereditary deafness. The differentiation of pluripotent stem cells into cochlear CX26-GJP-forming cells has not been reported. Here, we detail the development of a novel strategy to differentiate induced pluripotent stem cells into functional CX26-GJP-forming cells that exhibit spontaneous ATP- and hemichannel-mediated Ca²⁺ transients typical of the developing cochlea. Furthermore, these cells from CX26-deficient mice recapitulated the drastic disruption of GJPs, the primary pathology of GJB2-related hearing loss. These in vitro models should be useful for establishing inner-ear cell therapies and drug screening that target GJB2-related hearing loss.

SUBMITTER: Fukunaga I

PROVIDER: S-EPMC5161531 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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In Vitro Models of GJB2-Related Hearing Loss Recapitulate Ca<sup>2+</sup> Transients via a Gap Junction Characteristic of Developing Cochlea.

Fukunaga Ichiro I Fujimoto Ayumi A Hatakeyama Kaori K Aoki Toru T Nishikawa Atena A Noda Tetsuo T Minowa Osamu O Kurebayashi Nagomi N Ikeda Katsuhisa K Kamiya Kazusaku K

Stem cell reports 20161110 6

Mutation of the Gap Junction Beta 2 gene (GJB2) encoding connexin 26 (CX26) is the most frequent cause of hereditary deafness worldwide and accounts for up to 50% of non-syndromic sensorineural hearing loss cases in some populations. Therefore, cochlear CX26-gap junction plaque (GJP)-forming cells such as cochlear supporting cells are thought to be the most important therapeutic target for the treatment of hereditary deafness. The differentiation of pluripotent stem cells into cochlear CX26-GJP- ...[more]

PMID: 27840044

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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.

Dataset Information

In Vitro Models of GJB2-Related Hearing Loss Recapitulate Ca²⁺ Transients via a Gap Junction Characteristic of Developing Cochlea.

Publications

In Vitro Models of GJB2-Related Hearing Loss Recapitulate Ca<sup>2+</sup> Transients via a Gap Junction Characteristic of Developing Cochlea.

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Dataset Information

In Vitro Models of GJB2-Related Hearing Loss Recapitulate Ca2+ Transients via a Gap Junction Characteristic of Developing Cochlea.

Publications

In Vitro Models of GJB2-Related Hearing Loss Recapitulate Ca<sup>2+</sup> Transients via a Gap Junction Characteristic of Developing Cochlea.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

In Vitro Models of GJB2-Related Hearing Loss Recapitulate Ca²⁺ Transients via a Gap Junction Characteristic of Developing Cochlea.