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The D50N mutation and syndromic deafness: altered Cx26 hemichannel properties caused by effects on the pore and intersubunit interactions.

ABSTRACT: Mutations in the GJB2 gene, which encodes Cx26, are the most common cause of sensorineural deafness. In syndromic cases, such as keratitis-ichthyosis-deafness (KID) syndrome, in which deafness is accompanied by corneal inflammation and hyperkeratotic skin, aberrant hemichannel function has emerged as the leading contributing factor. We found that D50N, the most frequent mutation associated with KID syndrome, produces multiple aberrant hemichannel properties, including loss of inhibition by extracellular Ca(2+), decreased unitary conductance, increased open hemichannel current rectification and voltage-shifted activation. We demonstrate that D50 is a pore-lining residue and that negative charge at this position strongly influences open hemichannel properties. Examination of two putative intersubunit interactions involving D50 suggested by the Cx26 crystal structure, K61-D50 and Q48-D50, showed no evidence of a K61-D50 interaction in hemichannels. However, our data suggest that Q48 and D50 interact and disruption of this interaction shifts hemichannel activation positive along the voltage axis. Additional shifts in activation by extracellular Ca(2+) remained in the absence of a D50-Q48 interaction but required an Asp or Glu at position 50, suggesting a separate electrostatic mechanism that critically involves this position. In gap junction (GJ) channels, D50 substitutions produced loss of function, whereas K61 substitutions functioned as GJ channels but not as hemichannels. These data demonstrate that D50 exerts effects on Cx26 hemichannel and GJ channel function as a result of its dual role as a pore residue and a component of an intersubunit complex in the extracellular region of the hemichannel. Differences in the effects of substitutions in GJ channels and hemichannels suggest that perturbations in structure occur upon hemichannel docking that significantly impact function. Collectively, these data provide insight into Cx26 structure-function and the underlying bases for the phenotypes associated with KID syndrome patients carrying the D50N mutation.

SUBMITTER: Sanchez HA

PROVIDER: S-EPMC3691445 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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The D50N mutation and syndromic deafness: altered Cx26 hemichannel properties caused by effects on the pore and intersubunit interactions.

Sanchez Helmuth A HA Villone Krista K Srinivas Miduturu M Verselis Vytas K VK

The Journal of general physiology 20130701 1

Mutations in the GJB2 gene, which encodes Cx26, are the most common cause of sensorineural deafness. In syndromic cases, such as keratitis-ichthyosis-deafness (KID) syndrome, in which deafness is accompanied by corneal inflammation and hyperkeratotic skin, aberrant hemichannel function has emerged as the leading contributing factor. We found that D50N, the most frequent mutation associated with KID syndrome, produces multiple aberrant hemichannel properties, including loss of inhibition by extra ...[more]

PMID: 23797419

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Project description:Connexin 26 (Cx26) is a transmembrane protein that forms hexameric hemichannels that can function when unopposed or dock to form intercellular gap junction channels. Aberrantly functioning unopposed hemichannels are a common feature of syndromic deafness associated with mutations in Cx26. In this study, we examine two different mutations at the same position in the N-terminal domain of Cx26, N14K and N14Y, which have been reported to produce different phenotypes in patients. We find that both N14K and N14Y, when expressed alone or together with wild-type (WT) Cx26, result in functional hemichannels with widely disparate functional properties. N14K currents are robust, whereas N14Y currents are small. The two mutants also exhibit opposite shifts in voltage-dependent loop gating, such that activation of N14K and N14Y is shifted in the hyperpolarizing and depolarizing directions, respectively. Deactivation kinetics suggests that N14K stabilizes and N14Y destabilizes the open state. Single N14K hemichannel recordings in low extracellular Ca(2+) show no evidence of stable closing transitions associated with loop gating, and N14K hemichannels are insensitive to pH. Together, these properties cause N14K hemichannels to be particularly refractory to closing. Although we find that the unitary conductance of N14K is indistinguishable from WT Cx26, mutagenesis and substituted cysteine accessibility studies suggest that the N14 residue is exposed to the pore and that the differential properties of N14K and N14Y hemichannels likely result from altered electrostatic interactions between the N terminus and the cytoplasmic extension of TM2 in the adjacent subunit. The combined effects that we observe on loop gating and pH regulation may explain the unusual buccal cutaneous manifestations in patients carrying the N14K mutation. Our work also provides new considerations regarding the underlying molecular mechanism of loop gating, which controls hemichannel opening in the plasma membrane.

Dataset Information

The D50N mutation and syndromic deafness: altered Cx26 hemichannel properties caused by effects on the pore and intersubunit interactions.

Publications

The D50N mutation and syndromic deafness: altered Cx26 hemichannel properties caused by effects on the pore and intersubunit interactions.

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