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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: 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.

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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Extracellular zinc (Zn++) reduced hemichannel currents mediated by KID-causing Cx26 mutations(a) Representative current (Im) traces corresponding to a single Cx26-G45E-expressing cell recorded in the presence of 0, 10, and 100µM Zn++. An h2O-injected control cell is shown for comparison, (b) Mean currents plotted against membrane potential (Vm) illustrated current-voltage relationships. Control cells showed negligible current (N=10). Whole-cell currents observed in Cx26-G45E oocytes (N=16) were inhibited by addition of 1 (N=5), 10 (N=5), and 100µM (N=5) Zn++ to the medium, (c) Concentration dependent effects of zinc perfusion in cells expressing Cx26-G45E, -D50N, -A40V, -N14K, -G12R, -A88V, and -D50A. Bars represent the mean current as a percentage of the pre-drug value for five cells. Data are means ± SEM.
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Figure 4: Extracellular zinc (Zn++) reduced hemichannel currents mediated by KID-causing Cx26 mutations(a) Representative current (Im) traces corresponding to a single Cx26-G45E-expressing cell recorded in the presence of 0, 10, and 100µM Zn++. An h2O-injected control cell is shown for comparison, (b) Mean currents plotted against membrane potential (Vm) illustrated current-voltage relationships. Control cells showed negligible current (N=10). Whole-cell currents observed in Cx26-G45E oocytes (N=16) were inhibited by addition of 1 (N=5), 10 (N=5), and 100µM (N=5) Zn++ to the medium, (c) Concentration dependent effects of zinc perfusion in cells expressing Cx26-G45E, -D50N, -A40V, -N14K, -G12R, -A88V, and -D50A. Bars represent the mean current as a percentage of the pre-drug value for five cells. Data are means ± SEM.

Mentions: A limitation of quinine-family connexin inhibitors is their failure to distinguish between junctional and non-junctional channel configurations. Divalent cations inhibit connexin channels and have been shown to act at the extracellular aspect of the pore to promote loop gating (Verselis and Srinivas, 2008). Robust gap-junctional conductances are routinely measured from cell pairs in the presence of extracellular Ca++, indicating that binding of ions likely occurs at sites only accessible in undocked hemichannels. We recorded hemichannel currents from single Xenopus oocytes expressing Cx26-G45E in the presence and absence of 1,10, and 100µM extracellular Zn++. Oocytes injected with H2O passed negligible current, again providing a negative control. Those expressing Cx26-G45E showed large fluxes, as previously documented. Addition of Zn++ to the extracellular milieu caused membrane currents to diminish in a dose-dependent manner (figure 4a). Mean currents were plotted as a function of membrane potential for each recording condition to facilitate comparison of current-voltage relationships (figure 4b). Massive outward currents associated with Cx26-G45E were progressively reduced with 1,10, and 100µM Zn++ at all tested voltages. The degree of inhibition was quantified by perfusion of single cells during a paradigm of serial +100mV pulses. For cells expressing Cx26-G45E, 73±2.6% (N=5), 29±2.1% (N=5), and 12±3.9% (N=5) of the initial current persisted after 1.5min of 1,10, and 100µM Zn++, respectively (figure 4c, extrapolated IC50≈3µM).


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)

Extracellular zinc (Zn++) reduced hemichannel currents mediated by KID-causing Cx26 mutations(a) Representative current (Im) traces corresponding to a single Cx26-G45E-expressing cell recorded in the presence of 0, 10, and 100µM Zn++. An h2O-injected control cell is shown for comparison, (b) Mean currents plotted against membrane potential (Vm) illustrated current-voltage relationships. Control cells showed negligible current (N=10). Whole-cell currents observed in Cx26-G45E oocytes (N=16) were inhibited by addition of 1 (N=5), 10 (N=5), and 100µM (N=5) Zn++ to the medium, (c) Concentration dependent effects of zinc perfusion in cells expressing Cx26-G45E, -D50N, -A40V, -N14K, -G12R, -A88V, and -D50A. Bars represent the mean current as a percentage of the pre-drug value for five cells. Data are means ± SEM.
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Related In: Results  -  Collection

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Figure 4: Extracellular zinc (Zn++) reduced hemichannel currents mediated by KID-causing Cx26 mutations(a) Representative current (Im) traces corresponding to a single Cx26-G45E-expressing cell recorded in the presence of 0, 10, and 100µM Zn++. An h2O-injected control cell is shown for comparison, (b) Mean currents plotted against membrane potential (Vm) illustrated current-voltage relationships. Control cells showed negligible current (N=10). Whole-cell currents observed in Cx26-G45E oocytes (N=16) were inhibited by addition of 1 (N=5), 10 (N=5), and 100µM (N=5) Zn++ to the medium, (c) Concentration dependent effects of zinc perfusion in cells expressing Cx26-G45E, -D50N, -A40V, -N14K, -G12R, -A88V, and -D50A. Bars represent the mean current as a percentage of the pre-drug value for five cells. Data are means ± SEM.
Mentions: A limitation of quinine-family connexin inhibitors is their failure to distinguish between junctional and non-junctional channel configurations. Divalent cations inhibit connexin channels and have been shown to act at the extracellular aspect of the pore to promote loop gating (Verselis and Srinivas, 2008). Robust gap-junctional conductances are routinely measured from cell pairs in the presence of extracellular Ca++, indicating that binding of ions likely occurs at sites only accessible in undocked hemichannels. We recorded hemichannel currents from single Xenopus oocytes expressing Cx26-G45E in the presence and absence of 1,10, and 100µM extracellular Zn++. Oocytes injected with H2O passed negligible current, again providing a negative control. Those expressing Cx26-G45E showed large fluxes, as previously documented. Addition of Zn++ to the extracellular milieu caused membrane currents to diminish in a dose-dependent manner (figure 4a). Mean currents were plotted as a function of membrane potential for each recording condition to facilitate comparison of current-voltage relationships (figure 4b). Massive outward currents associated with Cx26-G45E were progressively reduced with 1,10, and 100µM Zn++ at all tested voltages. The degree of inhibition was quantified by perfusion of single cells during a paradigm of serial +100mV pulses. For cells expressing Cx26-G45E, 73±2.6% (N=5), 29±2.1% (N=5), and 12±3.9% (N=5) of the initial current persisted after 1.5min of 1,10, and 100µM Zn++, respectively (figure 4c, extrapolated IC50≈3µM).

Bottom Line: 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.

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