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.

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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)
National Institute of General Medical Sciences (NIGMS)
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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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Srinivas, Miduturu; Verselis, Vytas K; White, Thomas W (2018) Human diseases associated with connexin mutations. Biochim Biophys Acta Biomembr 1860:192-201
Verselis, Vytas K (2017) Connexin hemichannels and cochlear function. Neurosci Lett :
Sanchez, Helmuth A; Slavi, Nefeli; Srinivas, Miduturu et al. (2016) Syndromic deafness mutations at Asn 14 differentially alter the open stability of Cx26 hemichannels. J Gen Physiol 148:25-42
Sanchez, Helmuth A; Verselis, Vytas K (2014) Aberrant Cx26 hemichannels and keratitis-ichthyosis-deafness syndrome: insights into syndromic hearing loss. Front Cell Neurosci 8:354
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
Sanchez, Helmuth A; Villone, Krista; Srinivas, Miduturu et al. (2013) The D50N mutation and syndromic deafness: altered Cx26 hemichannel properties caused by effects on the pore and intersubunit interactions. J Gen Physiol 142:3-22
Verselis, Vytas K; Srinivas, Miduturu (2013) Connexin channel modulators and their mechanisms of action. Neuropharmacology 75:517-24
Kronengold, Jack; Srinivas, Miduturu; Verselis, Vytas K (2012) The N-terminal half of the connexin protein contains the core elements of the pore and voltage gates. J Membr Biol 245:453-63
Rubinos, Clio; Sánchez, 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
Sánchez, Helmuth A; Mese, Gülistan; 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

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