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Aberrant connexin26 hemichannels underlying keratitis-ichthyosis-deafness syndrome are potently inhibited by mefloquine.

Levit NA, Sellitto C, Wang HZ, Li L, Srinivas M, Brink PR, White TW - J. Invest. Dermatol. (2014)

Bottom Line: Functional analyses have revealed that a majority of KID-causing mutations confer a novel expansion of hemichannel activity, mediated by connexin channels in a nonjunctional configuration.Inappropriate Cx26 hemichannel opening is hypothesized to compromise keratinocyte integrity and epidermal homeostasis.Furthermore, we used freshly isolated transgenic keratinocytes to show that micromolar concentrations of MFQ attenuated increased macroscopic membrane currents in primary mouse keratinocytes expressing human Cx26-G45E, a mutation that causes a lethal form of KID syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York, USA.

ABSTRACT
Keratitis-ichthyosis-deafness (KID) syndrome is an ectodermal dysplasia caused by dominant mutations of connexin26 (Cx26). Loss of Cx26 function causes nonsyndromic sensorineural deafness, without consequence in the epidermis. Functional analyses have revealed that a majority of KID-causing mutations confer a novel expansion of hemichannel activity, mediated by connexin channels in a nonjunctional configuration. Inappropriate Cx26 hemichannel opening is hypothesized to compromise keratinocyte integrity and epidermal homeostasis. Pharmacological modulators of Cx26 are needed to assess the pathomechanistic involvement of hemichannels in the development of hyperkeratosis in KID syndrome. We have used electrophysiological assays to evaluate small-molecule analogs of quinine for suppressive effects on aberrant hemichannel currents elicited by KID mutations. Here, we show that mefloquine (MFQ) inhibits several mutant hemichannel forms implicated in KID syndrome when expressed in Xenopus laevis oocytes (IC50∼16 μM), using an extracellular divalent cation, zinc (Zn(++)), as a nonspecific positive control for comparison (IC50∼3 μM). Furthermore, we used freshly isolated transgenic keratinocytes to show that micromolar concentrations of MFQ attenuated increased macroscopic membrane currents in primary mouse keratinocytes expressing human Cx26-G45E, a mutation that causes a lethal form of KID syndrome.

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Cx26 mutations induced large hemichannel currents in Xenopus oocytes(a) Cells were clamped at −40mV and subjected to voltage pulses spanning −30 to +60mV in 10mV steps (Vm). h2O-injected cells displayed negligible whole-cell membrane currents (Im). Cx26 expressing oocytes all exhibited hemichannel currents, however, KID syndrome mutations showed much larger currents than wild-type (WT). (b) WT and mutant connexins are equivalently translated in Xenopus oocytes. Membrane extracts were probed with an antibody against Cx26. H2O-injected controls did not express Cx26, whereas WT, Cx26-G45E, -D50N, -A40V, -A88V, -D50A, -N14K, and -G12R were detected. Blots were also probed with an antibody against β-actin, the normalized ratio of Cx26 to β-actin expression was quantified, and found to be within ±10% of WT Cx26 for all mutations.
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Figure 1: Cx26 mutations induced large hemichannel currents in Xenopus oocytes(a) Cells were clamped at −40mV and subjected to voltage pulses spanning −30 to +60mV in 10mV steps (Vm). h2O-injected cells displayed negligible whole-cell membrane currents (Im). Cx26 expressing oocytes all exhibited hemichannel currents, however, KID syndrome mutations showed much larger currents than wild-type (WT). (b) WT and mutant connexins are equivalently translated in Xenopus oocytes. Membrane extracts were probed with an antibody against Cx26. H2O-injected controls did not express Cx26, whereas WT, Cx26-G45E, -D50N, -A40V, -A88V, -D50A, -N14K, and -G12R were detected. Blots were also probed with an antibody against β-actin, the normalized ratio of Cx26 to β-actin expression was quantified, and found to be within ±10% of WT Cx26 for all mutations.

Mentions: Previous reports have described increases in hemichannel functionality as a common feature shared by Cx26 mutations linked to KID syndrome (Donnelly etal., 2012; Gerido et al., 2007; Lee et al., 2009; Mhaske et al., 2013; Montgomery et al., 2004; Sanchez et al., 2010; Stong et al., 2006). Prior to pursuing inhibitor studies, we quantified membrane currents in single Xenopus oocytes expressing Cx26-G45E, -D50N, -A40V, -N14K, -G12R, -D50A, and -A88V, with Cx26-WT- and water-injected control cells. KID syndrome mutations result from single amino-acid substitutions that localize to the Cx26 N-terminus and first extracellular loop, with the exception of A88V, which appears in the second transmembrane domain. To assay membrane current, cells were voltage-clamped at −40mV and subjected to a series of depolarizing transmembrane voltages (figure 1a). Negligible membrane current was recorded from oocytes injected with H2O for voltages between −30 and +60mV. Wild-type Cx26 hemichannels favored a low open-probability resting state with outward current induced by membrane depolarization and an approximately linear current-voltage relationship as previously demonstrated (Gonzalez etal., 2006; Ripps etal., 2004). The Cx26-G45E, -D50N, -A40V, -N14K, -G12R, -D50A, and -A88V mutants displayed increased outward currents relative to H2O or Cx26-WT-injected cells. At the largest voltage tested, Cx26-WT hemichannels passed maximal currents of 0.5–1.5µA and recorded conductance was 10.5-, 7.5-, 8-, 8-, 4.5-, 4-, and 8-fold higher in Cx26-G45E, -D50N, -A40V, -N14K, -G12R, -D50A, and -A88V respectively. Western blotting of cell lysates for total Cx26 content eliminated the possibility that the different magnitudes of membrane current arose from unequal levels of protein expression (figure 1b). The Cx26 band intensity was approximately equal across the 7 mutant groups, and within ±10% of the expression level of Cx26-WT when normalized to β-actin.


Aberrant connexin26 hemichannels underlying keratitis-ichthyosis-deafness syndrome are potently inhibited by mefloquine.

Levit NA, Sellitto C, Wang HZ, Li L, Srinivas M, Brink PR, White TW - J. Invest. Dermatol. (2014)

Cx26 mutations induced large hemichannel currents in Xenopus oocytes(a) Cells were clamped at −40mV and subjected to voltage pulses spanning −30 to +60mV in 10mV steps (Vm). h2O-injected cells displayed negligible whole-cell membrane currents (Im). Cx26 expressing oocytes all exhibited hemichannel currents, however, KID syndrome mutations showed much larger currents than wild-type (WT). (b) WT and mutant connexins are equivalently translated in Xenopus oocytes. Membrane extracts were probed with an antibody against Cx26. H2O-injected controls did not express Cx26, whereas WT, Cx26-G45E, -D50N, -A40V, -A88V, -D50A, -N14K, and -G12R were detected. Blots were also probed with an antibody against β-actin, the normalized ratio of Cx26 to β-actin expression was quantified, and found to be within ±10% of WT Cx26 for all mutations.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4363291&req=5

Figure 1: Cx26 mutations induced large hemichannel currents in Xenopus oocytes(a) Cells were clamped at −40mV and subjected to voltage pulses spanning −30 to +60mV in 10mV steps (Vm). h2O-injected cells displayed negligible whole-cell membrane currents (Im). Cx26 expressing oocytes all exhibited hemichannel currents, however, KID syndrome mutations showed much larger currents than wild-type (WT). (b) WT and mutant connexins are equivalently translated in Xenopus oocytes. Membrane extracts were probed with an antibody against Cx26. H2O-injected controls did not express Cx26, whereas WT, Cx26-G45E, -D50N, -A40V, -A88V, -D50A, -N14K, and -G12R were detected. Blots were also probed with an antibody against β-actin, the normalized ratio of Cx26 to β-actin expression was quantified, and found to be within ±10% of WT Cx26 for all mutations.
Mentions: Previous reports have described increases in hemichannel functionality as a common feature shared by Cx26 mutations linked to KID syndrome (Donnelly etal., 2012; Gerido et al., 2007; Lee et al., 2009; Mhaske et al., 2013; Montgomery et al., 2004; Sanchez et al., 2010; Stong et al., 2006). Prior to pursuing inhibitor studies, we quantified membrane currents in single Xenopus oocytes expressing Cx26-G45E, -D50N, -A40V, -N14K, -G12R, -D50A, and -A88V, with Cx26-WT- and water-injected control cells. KID syndrome mutations result from single amino-acid substitutions that localize to the Cx26 N-terminus and first extracellular loop, with the exception of A88V, which appears in the second transmembrane domain. To assay membrane current, cells were voltage-clamped at −40mV and subjected to a series of depolarizing transmembrane voltages (figure 1a). Negligible membrane current was recorded from oocytes injected with H2O for voltages between −30 and +60mV. Wild-type Cx26 hemichannels favored a low open-probability resting state with outward current induced by membrane depolarization and an approximately linear current-voltage relationship as previously demonstrated (Gonzalez etal., 2006; Ripps etal., 2004). The Cx26-G45E, -D50N, -A40V, -N14K, -G12R, -D50A, and -A88V mutants displayed increased outward currents relative to H2O or Cx26-WT-injected cells. At the largest voltage tested, Cx26-WT hemichannels passed maximal currents of 0.5–1.5µA and recorded conductance was 10.5-, 7.5-, 8-, 8-, 4.5-, 4-, and 8-fold higher in Cx26-G45E, -D50N, -A40V, -N14K, -G12R, -D50A, and -A88V respectively. Western blotting of cell lysates for total Cx26 content eliminated the possibility that the different magnitudes of membrane current arose from unequal levels of protein expression (figure 1b). The Cx26 band intensity was approximately equal across the 7 mutant groups, and within ±10% of the expression level of Cx26-WT when normalized to β-actin.

Bottom Line: Functional analyses have revealed that a majority of KID-causing mutations confer a novel expansion of hemichannel activity, mediated by connexin channels in a nonjunctional configuration.Inappropriate Cx26 hemichannel opening is hypothesized to compromise keratinocyte integrity and epidermal homeostasis.Furthermore, we used freshly isolated transgenic keratinocytes to show that micromolar concentrations of MFQ attenuated increased macroscopic membrane currents in primary mouse keratinocytes expressing human Cx26-G45E, a mutation that causes a lethal form of KID syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York, USA.

ABSTRACT
Keratitis-ichthyosis-deafness (KID) syndrome is an ectodermal dysplasia caused by dominant mutations of connexin26 (Cx26). Loss of Cx26 function causes nonsyndromic sensorineural deafness, without consequence in the epidermis. Functional analyses have revealed that a majority of KID-causing mutations confer a novel expansion of hemichannel activity, mediated by connexin channels in a nonjunctional configuration. Inappropriate Cx26 hemichannel opening is hypothesized to compromise keratinocyte integrity and epidermal homeostasis. Pharmacological modulators of Cx26 are needed to assess the pathomechanistic involvement of hemichannels in the development of hyperkeratosis in KID syndrome. We have used electrophysiological assays to evaluate small-molecule analogs of quinine for suppressive effects on aberrant hemichannel currents elicited by KID mutations. Here, we show that mefloquine (MFQ) inhibits several mutant hemichannel forms implicated in KID syndrome when expressed in Xenopus laevis oocytes (IC50∼16 μM), using an extracellular divalent cation, zinc (Zn(++)), as a nonspecific positive control for comparison (IC50∼3 μM). Furthermore, we used freshly isolated transgenic keratinocytes to show that micromolar concentrations of MFQ attenuated increased macroscopic membrane currents in primary mouse keratinocytes expressing human Cx26-G45E, a mutation that causes a lethal form of KID syndrome.

Show MeSH
Related in: MedlinePlus