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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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Mefloquine (MFQ) and quinine derivatives (QUO) suppressed (a) Cx26-G45E and (b) -D50N hemichannel currents in Xenopus oocytesSingle cells held at −40mV 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.
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Figure 2: Mefloquine (MFQ) and quinine derivatives (QUO) suppressed (a) Cx26-G45E and (b) -D50N hemichannel currents in Xenopus oocytesSingle cells held at −40mV 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.

Mentions: Molecules therapeutically classified as antimalarial agents have been recognized to suppress hemichannel currents by direct action on connexin subunits and partial-selectivity properties are conferred to these compounds by differences in affinities for connexin subtypes (Cruikshank et al., 2004; Rubinos et al., 2012; Srinivas et al., 2001). Inhibitor studies involving mefloquine have focused on connexin50 and connexin36, in the context of their roles as gap-junction proteins that couple lens epithelial cells and neurons, respectively (Cruikshank et al., 2004). The ability of mefloquine to modulate connexin26 channel activity had received only cursory examination and only with regard to wild-type junctional communication. We screened mefloquine and four related derivatives (QU020, QU021, QU022, and QU026) for inhibitory capacity against dysregulated hemichannels resulting from two connexin26 mutations associated with KID syndrome. Cx26-G45E causes a lethal form of KID syndrome (Griffith etal., 2006; Janecke etal., 2005; Jonard etal., 2008) and is characterized by robust hemichannel activity that represents the most significant deviation from wild-type channel behavior (Gerido etal., 2007). Cx26-D50N is the most commonly reported mutation in cases of KID syndrome. Drug screening was performed by perfusion of candidate inhibitors during voltage-clamp recording of Cx26-G45E (figure 2a) and Cx26-D50N (figure 2b) hemichannel currents in single Xenopus oocytes. Sequential depolarizing +50mV pulses stimulated repeated channel opening and consistent bursts of whole-cell membrane current. Inhibitor effects were evaluated by exchange of the bathing media for a segment of each recording (figure 2, left). At a drug concentration of 30µM, QU022 displayed unimpressive inhibition of membrane currents (<20% reduction) for both Cx26-G45E and Cx26-D50N. QU022 lacks the aliphatic piperidine ring present in mefloquine and also substitutes a -CCI3 group for the -CF3 found on the quinolone ring, representing the most dissimilar molecule to mefloquine tested. QU020 also failed to produce any dramatic suppression of Cx26-G45E hemichannels (25±14%) but was twice as effective when tested on Cx26-D50N hemichannels (49±7.3%). QU021 performed at a similar level, approximately halving membrane currents passed by both mutant channels (52±7.8% and 43±12% for Cx26-G45E and -D50N respectively). Mefloquine and QU026 elicited the most striking diminution in membrane currents recorded from single cells expressing either Cx26-G45E (70±17% for 30µM MFQ; 59±13% for 30µM QU026) (figure 2a, right) or Cx26-D50N (69±15% for 30µM MFQ; 73±11% for 30µM QU026) (figure 2b, right). QU026 replaces the piperidine ring in mefloquine with a third aromatic ring but includes no other structural deviation, possibly accounting for the parallel results. Two -CF3 groups appear on the quinolone backbone of mefloquine, QU020, QU021, and QU026—a feature that enhances the lipophilicity of these molecules. For this reason, it is possible that lipid-rich yolk granules abundant in stage V–VI oocytes may sequester a portion of the drug, effectively reducing the delivered dose and causing underreporting of potency in this system. Lipophilicity is, however, an appealing property of any drug considered for targeting epidermal proteins via topical delivery strategies. Given the status of mefloquine as an FDA approved drug with a history of safety and pharmacokinetic data, it was selected for further characterization.


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)

Mefloquine (MFQ) and quinine derivatives (QUO) suppressed (a) Cx26-G45E and (b) -D50N hemichannel currents in Xenopus oocytesSingle cells held at −40mV 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.
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Related In: Results  -  Collection

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Figure 2: Mefloquine (MFQ) and quinine derivatives (QUO) suppressed (a) Cx26-G45E and (b) -D50N hemichannel currents in Xenopus oocytesSingle cells held at −40mV 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.
Mentions: Molecules therapeutically classified as antimalarial agents have been recognized to suppress hemichannel currents by direct action on connexin subunits and partial-selectivity properties are conferred to these compounds by differences in affinities for connexin subtypes (Cruikshank et al., 2004; Rubinos et al., 2012; Srinivas et al., 2001). Inhibitor studies involving mefloquine have focused on connexin50 and connexin36, in the context of their roles as gap-junction proteins that couple lens epithelial cells and neurons, respectively (Cruikshank et al., 2004). The ability of mefloquine to modulate connexin26 channel activity had received only cursory examination and only with regard to wild-type junctional communication. We screened mefloquine and four related derivatives (QU020, QU021, QU022, and QU026) for inhibitory capacity against dysregulated hemichannels resulting from two connexin26 mutations associated with KID syndrome. Cx26-G45E causes a lethal form of KID syndrome (Griffith etal., 2006; Janecke etal., 2005; Jonard etal., 2008) and is characterized by robust hemichannel activity that represents the most significant deviation from wild-type channel behavior (Gerido etal., 2007). Cx26-D50N is the most commonly reported mutation in cases of KID syndrome. Drug screening was performed by perfusion of candidate inhibitors during voltage-clamp recording of Cx26-G45E (figure 2a) and Cx26-D50N (figure 2b) hemichannel currents in single Xenopus oocytes. Sequential depolarizing +50mV pulses stimulated repeated channel opening and consistent bursts of whole-cell membrane current. Inhibitor effects were evaluated by exchange of the bathing media for a segment of each recording (figure 2, left). At a drug concentration of 30µM, QU022 displayed unimpressive inhibition of membrane currents (<20% reduction) for both Cx26-G45E and Cx26-D50N. QU022 lacks the aliphatic piperidine ring present in mefloquine and also substitutes a -CCI3 group for the -CF3 found on the quinolone ring, representing the most dissimilar molecule to mefloquine tested. QU020 also failed to produce any dramatic suppression of Cx26-G45E hemichannels (25±14%) but was twice as effective when tested on Cx26-D50N hemichannels (49±7.3%). QU021 performed at a similar level, approximately halving membrane currents passed by both mutant channels (52±7.8% and 43±12% for Cx26-G45E and -D50N respectively). Mefloquine and QU026 elicited the most striking diminution in membrane currents recorded from single cells expressing either Cx26-G45E (70±17% for 30µM MFQ; 59±13% for 30µM QU026) (figure 2a, right) or Cx26-D50N (69±15% for 30µM MFQ; 73±11% for 30µM QU026) (figure 2b, right). QU026 replaces the piperidine ring in mefloquine with a third aromatic ring but includes no other structural deviation, possibly accounting for the parallel results. Two -CF3 groups appear on the quinolone backbone of mefloquine, QU020, QU021, and QU026—a feature that enhances the lipophilicity of these molecules. For this reason, it is possible that lipid-rich yolk granules abundant in stage V–VI oocytes may sequester a portion of the drug, effectively reducing the delivered dose and causing underreporting of potency in this system. Lipophilicity is, however, an appealing property of any drug considered for targeting epidermal proteins via topical delivery strategies. Given the status of mefloquine as an FDA approved drug with a history of safety and pharmacokinetic data, it was selected for further characterization.

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