Application of SCAM (Substituted Cysteine Accessibility Method) to Gap Junction Intercellular Channels

Skerrett, I. Martha; Kasperek, Eileen M.; Cao, Fengli; Shin, Jae‐Gook; Aronowitz, J.; Ahmed, Saifuddin; Nicholson, Bruce J.

doi:10.3109/15419060109080720

Cited by 8 publications

(9 citation statements)

References 19 publications

(15 reference statements)

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“…Singlechannel analysis of Cx32M34T revealed that these channels reside predominantly in a low conductance state, similar to the residual conductance state associated with voltage gating (20). In addition, the M34T substitution in Cx32 has been shown to induce a constriction of the pore (19), further supporting the likelihood that these channels are only partially open at rest. However, single-channel analysis is ultimately required to determine whether Vj induces an increase in Po or γ for Cx26M34T.…”

Section: Gating Characteristics Of M34tmentioning

confidence: 95%

Aberrant gating, but a normal expression pattern, underlies the recessive phenotype of the deafness mutant Connexin26M34T

Skerrett¹,

Di²,

Kasperek³

et al. 2004

FASEB j.

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Mutations in the gene GJB2, encoding the gap junction protein Connexin26 (Cx26), are the most prevalent cause of inherited hearing loss, and Cx26M34T was one of the first mutations linked to deafness (Kelsell et al., 1997; Nature 387, 80-83). We report the first characterization of the gating properties of M34T, which had previously been reported to be nonfunctional. Although homotypic mutant channels did not produce detectable currents, heterotypic pairings with wtCx26 confirmed that M34T formed intercellular channels, although the gating properties were altered. Cx26M34T displayed an inverted response to transjunctional voltage (Vj), mediating currents that activate in a time- and Vj-dependent manner. These characteristics suggest that the channel population is only partially open at rest, consistent with previous reports that dye transfer in M34T-expressing cells is reduced or abolished (e.g., Thonnissen et al., Human Genet. 111, 190-197). To investigate the controversial recessive/dominant behavior of this mutant, we coexpressed M34T with wtCx26 RNA at equimolar levels, mimicking the situation in heterozygotic individuals. Under these conditions, M34T did not significantly reduce Cx26/Cx26 coupling, or alter the electrophysiological properties of the wt channels, consistent with the recessive nature of the allele. Overexpression of the mutant did have some inhibitory effects on conductance, possibly explaining some of the previous reports in exogenous expression systems and some patients. Consistent with its electrophysiological behavior, we also show that M34T localizes to cell junctions in both transfected HeLa cells and patient-derived tissue.

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Section: Gating Characteristics Of M34tmentioning

confidence: 95%

Aberrant gating, but a normal expression pattern, underlies the recessive phenotype of the deafness mutant Connexin26M34T

Skerrett¹,

Di²,

Kasperek³

et al. 2004

FASEB j.

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“…These structural studies, however, have not provided unambiguous information on helix packing, the residues that form the pore, or the structure of the regulatory C-terminal domain. Studies in which native residues were substituted with Cys and then reacted with thiol reagents, however, suggest that transmembrane segments 1, 2, and 3 (M1-M3) are the helices lining the pore (23)(24)(25). These studies show the power of the combination of biochemical and functional studies to address specific structural aspects of connexins.…”

mentioning

confidence: 99%

Functional Expression in Xenopus Oocytes of Gap-junctional Hemichannels Formed by a Cysteine-less Connexin 43

Bao¹,

Chen

Reuss

et al. 2004

Journal of Biological Chemistry

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Gap-junctional channels are formed by two connexons or gap-junctional hemichannels in series, with each connexon conformed by six connexin molecules. As with other membrane proteins, structural information on connexons can potentially be obtained with techniques that take advantage of the highly specific thiol chemistry by positioning Cys residues at locations of interest, ideally in an otherwise Cys-less protein. It has been shown that conserved Cys residues located in the extracellular loops of connexins are essential for the docking of connexons from adjacent cells, preventing the formation of functional gap-junctional channels. Here we engineered a Cys-less version of connexin 43 (Cx43) and assessed its function using a Xenopus oocyte expression system. The Cys-less protein was expressed at the plasma membrane and did not form gap-junctional channels but formed hemichannels that behave similarly to those formed by Cx43 in terms of permeation to carboxyfluorescein. The carboxyfluorescein permeability of Cys-less hemichannels was increased by protein kinase C inhibition, like the wild-type Cx43 hemichannels. We generated a protein with a single Cys in a position (residue 34) thought to face the channel pore and show that thiol modification of the Cys abolishes the carboxyfluorescein permeability. We conclude that Cysless Cx43 forms regulated functional hemichannels, and therefore Cys-less Cx43 is a useful tool for future structural studies.Gap junctions are aqueous channels that connect neighboring cells electrically and chemically by mediating permeation of inorganic ions and solutes of M r Ͻ 1000 (Ref. 1 and reviewed in Ref.2). They are formed by two connexons or gap-junctional hemichannels in series with each connexon conformed by six connexin molecules (3-5). In addition, gap-junctional hemichannels have been identified in the plasma membrane of native cells and cell lines (6 -8) where they appear to participate in a number of physiological and pathophysiological processes (6 -15). Because of the roles of gap-junctional channels in development, organ function, and disease processes and the possible physiological and pathophysiological roles of gap-junctional hemichannels (see Ref. 2), there is considerable interest in understanding the structure and regulation of connexons. Despite all the effort devoted to study connexins, basic aspects of the structure and function of the protein remain unknown.The connexin 43 isoform (Cx43) 1 is expressed in organs such as the brain, myocardium, and kidney as well as in capillary endothelial cells (16 -22). A medium resolution structure of this connexin, without the C-terminal domain, has been solved by cryoelectron microscopy of two-dimensional crystals (4, 5). These structural studies, however, have not provided unambiguous information on helix packing, the residues that form the pore, or the structure of the regulatory C-terminal domain. Studies in which native residues were substituted with Cys and then reacted with thiol reagents, however, suggest that transmembran...

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“…The inner diameter of the membrane-embedded portion of the connexon pore narrowed from 40 to 14 Å due to a kink in TM1 followed by a 3 10 helix. TM1 was identified as the primary pore-lining domain, as previously suggested by Verselis et al, 2009, resolving conflicting results from molecular modeling, Cys-scanning, and domain-swapping studies (Fleishman et al, 2004; Skerrett et al, 2001; Zhou et al, 1997; Kronengold et al, 2003). However, the resolution did not permit side chain placement.…”

Section: Gap Junction Structure: High Resolutionmentioning

confidence: 68%

A History of Gap Junction Structure: Hexagonal Arrays to Atomic Resolution

Grosely

Sorgen

2013

Cell Communication & Adhesion

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Gap junctions are specialized membrane structures that provide an intercellular pathway for the propagation and/or amplification of signaling cascades responsible for impulse propagation, cell growth, and development. Prior to the identification of the proteins that comprise gap junctions, elucidation of channel structure began with initial observations of a hexagonal nexus connecting apposed cellular membranes. Concomitant with technological advancements spanning over 50 years, atomic resolution structures are now available detailing channel architecture and the cytoplasmic domains that have helped to define mechanisms governing the regulation of gap junctions. Highlighted in this review are the seminal structural studies that have led to our current understanding of gap junction biology.

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Application of SCAM (Substituted Cysteine Accessibility Method) to Gap Junction Intercellular Channels

Cited by 8 publications

References 19 publications

Aberrant gating, but a normal expression pattern, underlies the recessive phenotype of the deafness mutant Connexin26M34T

Aberrant gating, but a normal expression pattern, underlies the recessive phenotype of the deafness mutant Connexin26M34T

Functional Expression in Xenopus Oocytes of Gap-junctional Hemichannels Formed by a Cysteine-less Connexin 43

A History of Gap Junction Structure: Hexagonal Arrays to Atomic Resolution

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