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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
.
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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.
Figure 2. Mefloquine (MFQ) and quinine derivatives (QUO) suppressed (a) Cx26-G45E and (b) -D50N hemichannel currents in Xenopus oocytesSingle cells held at â40mV were repeatedly pulsed with +50mV depolarizations (Vm) and membrane current (Im) was measured. Cells were exposed to 30µM inhibitor for 90sec by switching perfusion solutions after lmin (left, shown for mefloquine). Inhibitors were washed out for 2.5min, showing partial reversibility at the concentration tested. Summary data for inhibitors QU020, QU021, QU022, MFQ, and QU026 are shown as the mean residual instantaneous membrane current during 30µM drug application as a percentage of the pre-drug value (right). MFQ and QU026 produced the greatest inhibition of Cx26-G45E and -D50N membrane currents. Data are the means ± SD.
Figure 3. Mefloquine (MFQ) attenuated KID-associated Cx26 hemichannel currents in a concentration-dependent and mutant-selective manner(a) Voltage-clamp recordings for three cells expressing Cx26-G45E showed an increasing magnitude of membrane current (Im) inhibition with 10, 30, and 100µM MFQ. (b) Mean MFQ response characteristics across Cx26-G45E-expressing cells showed that membrane current, plotted as a fraction of the starting value, fell by >25%, >50%, and >75% upon exposure to 10 (N=5), 30 (N=5), and l00µM (N=10) MFQ, respectively, (c) Effect of 10, 30, and 100µM MFQ perfusion on cells injected with Cx26-D50N, -A40V, -N14K, -G12R, -A88V, and -D50A. Bars represent the mean membrane current in the presence of the inhibitor as a percentage of the pre-drug value (n=5). Data are means ± SEM.
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.
Figure 5. Mefloquine inhibited hemichannel activity in transgenic Cx26-G45E primary keratinocytes(aâb) Cx26-G45E mice recapitulate the epidermal pathology of KID syndrome using inducible tissue-specific expression of Cx26-G45E and eGFP in a hairless background, (c) In vivo fluorescence imaging demonstrated spatial correlation of fluorescence with skin lesions, (d) Keratinocytes isolated from Cx26-G45E lesions retained eGFP expression, facilitating their identification for patch-clamp electrophysiology (bar, 10µm). (e) Whole-cell membrane currents (Im) were recorded by patch-clamp electrophysiology. Nominal currents were observed in control keratinocytes. (f) Current density plotted against membrane potential (Vm) showed large macroscopic currents were elicited from Cx26-G45E cells (N=9) that were diminished by the addition of 100µM mefloquine (N=9) to levels resembling control cells (N=5). Data are means ± SEM.
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