Mutations in the GJB2 gene that encodes the Cx26 gap junction (GJ) protein are one of the most common causes of inherited deafness in the human population. A subset of these mutations leads to syndromic forms of deafness in which sensorineural hearing loss is accompanied by severe, inflammatory skin disorders, such as keratitis-ichthiosis-deafness (KID) syndrome. The underlying basis of syndromic deafness appears to be aberrantly behaving hemichannels, a relatively new mechanism identified among Cx-related disorders. These hemichannels do not participate in the formation of intercellular GJ channels, but rather remain undocked and function as large, ion channels in the plasma membrane. Mutant hemichannels have been described to behave in a "leaky" manner leading to compromised cell function and cell death. In this proposal, we use a combination of molecular, biophysical and imaging approaches to investigate the mechanisms by which Cx hemichannels are dysfunctional in KID syndrome. The mutations notably cluster in two domains, the N- terminus (NT) and the first extracellular loop (E1), which we identified to be principal components of the Cx channel pore and to play essential roles in Cx hemichannel gating by voltage and regulation by extracellular Ca2+. Armed with expertise in these regions and a recently published crystal structure of Cx26, specific models of inter-subunit interactions involving E1 and NT residues will be examined along with their role in hemichannel gating and regulation. We focus on regulation by extracellular Ca2+, which is the most prevalent dysfunctional characteristic of KID syndrome mutants. We investigate the mechanistic link between Ca2+, pH and a distinct form of voltage gating, we originally described and termed loop gating, which robustly regulates opening and closing of hemichannels. Regulation by pH has been largely overlooked as a contributing factor to KID syndrome, as has been hemichannel selectivity. Using cysteine-substitution accessibility, we established that two KID syndrome mutants, G45E and D50N, are pore-lining and will investigate whether permeabilities to the key signaling molecules ATP and Ca2+ are significantly altered. G45E leads to a particularly severe, often fatal form of KID syndrome and our initial studies suggest increased permeability to Ca2+, rather than dysregulated gating, may be the key contributing factor. We extend these studies to keratinocytes isolated from transgenic animals carrying a G45E mutation driven under an inducible keratinocyte-specific promoter. Finally, some KID mutants fail to function as GJs channels despite functioning as hemichannels and we will investigate whether these mutant hemichannels exhibit an impaired ability to dock. Together, these studies explore the mechanistic bases of hemichannel dysfunction in Cx26 that lead to severe disorders in humans. These studies should also shed light on a growing list of disorders ascribed to hemichannel dysfunction that includes atherosclerosis, stroke, neuropathy and congenital cataractogenesis and should lead to strategies for treatment.

Public Health Relevance

Mutations in the gene that encodes the connexin26 ion channel protein are one of the most common causes of inherited deafness in the human population. In addition, a subset of mutations cause severe skin disorders and predispose patients to skin cancer (squamous cell carcinoma) and increased incidence of bacterial, viral, and fungal infections. This proposal investigates the underlying mechanisms causing these disorders.

National Institute of Health (NIH)
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Special Emphasis Panel (ZRG1)
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Nie, Zhongzhen
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Albert Einstein College of Medicine
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Sanchez, Helmuth A; Bienkowski, Rick; Slavi, Nefeli et al. (2014) Altered inhibition of Cx26 hemichannels by pH and Zn2+ in the A40V mutation associated with keratitis-ichthyosis-deafness syndrome. J Biol Chem 289:21519-32
Verselis, Vytas K; Srinivas, Miduturu (2013) Connexin channel modulators and their mechanisms of action. Neuropharmacology 75:517-24
Rubinos, Clio; Sanchez, Helmuth A; Verselis, Vytas K et al. (2012) Mechanism of inhibition of connexin channels by the quinine derivative N-benzylquininium. J Gen Physiol 139:69-82
Sanchez, Helmuth A; Mese, Gulistan; Srinivas, Miduturu et al. (2010) Differentially altered Ca2+ regulation and Ca2+ permeability in Cx26 hemichannels formed by the A40V and G45E mutations that cause keratitis ichthyosis deafness syndrome. J Gen Physiol 136:47-62
Verselis, Vytas K; Trelles, Maria P; Rubinos, Clio et al. (2009) Loop gating of connexin hemichannels involves movement of pore-lining residues in the first extracellular loop domain. J Biol Chem 284:4484-93
Sanchez, Helmuth A; Orellana, Juan A; Verselis, Vytas K et al. (2009) Metabolic inhibition increases activity of connexin-32 hemichannels permeable to Ca2+ in transfected HeLa cells. Am J Physiol Cell Physiol 297:C665-78
Verselis, Vytas K; Srinivas, Miduturu (2008) Divalent cations regulate connexin hemichannels by modulating intrinsic voltage-dependent gating. J Gen Physiol 132:315-27
Rackauskas, Mindaugas; Verselis, Vytas K; Bukauskas, Feliksas F (2007) Permeability of homotypic and heterotypic gap junction channels formed of cardiac connexins mCx30.2, Cx40, Cx43, and Cx45. Am J Physiol Heart Circ Physiol 293:H1729-36
Bukauskas, Feliksas F; Kreuzberg, Maria M; Rackauskas, Mindaugas et al. (2006) Properties of mouse connexin 30.2 and human connexin 31.9 hemichannels: implications for atrioventricular conduction in the heart. Proc Natl Acad Sci U S A 103:9726-31
Srinivas, Miduturu; Calderon, D Paola; Kronengold, Jack et al. (2006) Regulation of connexin hemichannels by monovalent cations. J Gen Physiol 127:67-75

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