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Connexin26 hemichannels with a mutation that causes KID syndrome in humans lack sensitivity to CO2.

Meigh L, Hussain N, Mulkey DK, Dale N - Elife (2014)

Bottom Line: Previously we have shown that Cx26 hemichannels are directly opened by CO2 (Meigh et al., 2013).However the effects of human disease-causing mutations on the CO2 sensitivity of Cx26 are entirely unknown.Here, we report the first connection between the CO2 sensitivity of Cx26 and human pathology, by demonstrating that Cx26 hemichannels with the mutation A88V, linked to Keratitis-Ichthyosis-Deafness syndrome, are both CO2 insensitive and associated with disordered breathing in humans.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Warwick, Coventry, United Kingdom.

ABSTRACT
AbstractMutations in connexin26 (Cx26) underlie a range of serious human pathologies. Previously we have shown that Cx26 hemichannels are directly opened by CO2 (Meigh et al., 2013). However the effects of human disease-causing mutations on the CO2 sensitivity of Cx26 are entirely unknown. Here, we report the first connection between the CO2 sensitivity of Cx26 and human pathology, by demonstrating that Cx26 hemichannels with the mutation A88V, linked to Keratitis-Ichthyosis-Deafness syndrome, are both CO2 insensitive and associated with disordered breathing in humans.

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HeLa cells transfected with the empty pCAG-GS mCherry vector show no sensitivity to CO2 and do not dye load when exposed to zero Ca2+ aCSF.(A) Cumulative probability distributions of pixel intensity for HeLa cells transfected with pCAG-GS mCherry under control, hypercapnia and zero Ca2+ conditions. The cells were exposed to 200 µM CBF for 5 min under each condition before being washed. The graphs show all of the measurements from 4 independent repetitions for each condition. (B) When transfected with pCAG-GS mCherry, the HeLa cells exhibit diffuse red fluorescence from expression of the mCherry. This contrasts with the punctate fluorescence seen flowing transfection with pCAG-GS Cx26-mCherry (inset). Scale bars 20 µm.DOI:http://dx.doi.org/10.7554/eLife.04249.003
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fig2s1: HeLa cells transfected with the empty pCAG-GS mCherry vector show no sensitivity to CO2 and do not dye load when exposed to zero Ca2+ aCSF.(A) Cumulative probability distributions of pixel intensity for HeLa cells transfected with pCAG-GS mCherry under control, hypercapnia and zero Ca2+ conditions. The cells were exposed to 200 µM CBF for 5 min under each condition before being washed. The graphs show all of the measurements from 4 independent repetitions for each condition. (B) When transfected with pCAG-GS mCherry, the HeLa cells exhibit diffuse red fluorescence from expression of the mCherry. This contrasts with the punctate fluorescence seen flowing transfection with pCAG-GS Cx26-mCherry (inset). Scale bars 20 µm.DOI:http://dx.doi.org/10.7554/eLife.04249.003

Mentions: We introduced the A88V mutation into Cx26 and then tested the CO2 sensitivity of Cx26A88V hemichannels expressed in HeLa cells via an established and sensitive dye-loading protocol (Huckstepp et al., 2010a; Meigh et al., 2013). Under conditions of normal extracellular Ca2+, HeLa cells expressing wild type Cx26 hemichannels readily load with carboxyfluorescein when exposed to a moderately hypercapnic saline (PCO2 55 mmHg) (Huckstepp et al., 2010a; Meigh et al., 2013). However HeLa cells expressing Cx26A88V showed no such CO2-dependent dye loading even when exposed to higher levels of PCO2 (70 mmHg, Figure 2). The failure to exhibit CO2-dependent dye loading was not due to a lack of functional hemichannels as the positive control of removing extracellular Ca2+, which opens all connexin hemichannels, caused robust dye loading (Figure 2). HeLa cells transfected with an empty vector do not show any dye loading in response to a CO2 stimulus or removal of extracellular Ca2+ (Figure 2—figure supplement 1). Surprisingly therefore, the conservative mutation A88V caused Cx26 hemichannels to lose their sensitivity to CO2. As this mutation is far from the residues involved in CO2 binding (K125 and R104), the mechanism for the loss of CO2 sensitivity is unclear.10.7554/eLife.04249.004Figure 2.Cx26A88V hemichannels are no longer sensitive to CO2.


Connexin26 hemichannels with a mutation that causes KID syndrome in humans lack sensitivity to CO2.

Meigh L, Hussain N, Mulkey DK, Dale N - Elife (2014)

HeLa cells transfected with the empty pCAG-GS mCherry vector show no sensitivity to CO2 and do not dye load when exposed to zero Ca2+ aCSF.(A) Cumulative probability distributions of pixel intensity for HeLa cells transfected with pCAG-GS mCherry under control, hypercapnia and zero Ca2+ conditions. The cells were exposed to 200 µM CBF for 5 min under each condition before being washed. The graphs show all of the measurements from 4 independent repetitions for each condition. (B) When transfected with pCAG-GS mCherry, the HeLa cells exhibit diffuse red fluorescence from expression of the mCherry. This contrasts with the punctate fluorescence seen flowing transfection with pCAG-GS Cx26-mCherry (inset). Scale bars 20 µm.DOI:http://dx.doi.org/10.7554/eLife.04249.003
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4270064&req=5

fig2s1: HeLa cells transfected with the empty pCAG-GS mCherry vector show no sensitivity to CO2 and do not dye load when exposed to zero Ca2+ aCSF.(A) Cumulative probability distributions of pixel intensity for HeLa cells transfected with pCAG-GS mCherry under control, hypercapnia and zero Ca2+ conditions. The cells were exposed to 200 µM CBF for 5 min under each condition before being washed. The graphs show all of the measurements from 4 independent repetitions for each condition. (B) When transfected with pCAG-GS mCherry, the HeLa cells exhibit diffuse red fluorescence from expression of the mCherry. This contrasts with the punctate fluorescence seen flowing transfection with pCAG-GS Cx26-mCherry (inset). Scale bars 20 µm.DOI:http://dx.doi.org/10.7554/eLife.04249.003
Mentions: We introduced the A88V mutation into Cx26 and then tested the CO2 sensitivity of Cx26A88V hemichannels expressed in HeLa cells via an established and sensitive dye-loading protocol (Huckstepp et al., 2010a; Meigh et al., 2013). Under conditions of normal extracellular Ca2+, HeLa cells expressing wild type Cx26 hemichannels readily load with carboxyfluorescein when exposed to a moderately hypercapnic saline (PCO2 55 mmHg) (Huckstepp et al., 2010a; Meigh et al., 2013). However HeLa cells expressing Cx26A88V showed no such CO2-dependent dye loading even when exposed to higher levels of PCO2 (70 mmHg, Figure 2). The failure to exhibit CO2-dependent dye loading was not due to a lack of functional hemichannels as the positive control of removing extracellular Ca2+, which opens all connexin hemichannels, caused robust dye loading (Figure 2). HeLa cells transfected with an empty vector do not show any dye loading in response to a CO2 stimulus or removal of extracellular Ca2+ (Figure 2—figure supplement 1). Surprisingly therefore, the conservative mutation A88V caused Cx26 hemichannels to lose their sensitivity to CO2. As this mutation is far from the residues involved in CO2 binding (K125 and R104), the mechanism for the loss of CO2 sensitivity is unclear.10.7554/eLife.04249.004Figure 2.Cx26A88V hemichannels are no longer sensitive to CO2.

Bottom Line: Previously we have shown that Cx26 hemichannels are directly opened by CO2 (Meigh et al., 2013).However the effects of human disease-causing mutations on the CO2 sensitivity of Cx26 are entirely unknown.Here, we report the first connection between the CO2 sensitivity of Cx26 and human pathology, by demonstrating that Cx26 hemichannels with the mutation A88V, linked to Keratitis-Ichthyosis-Deafness syndrome, are both CO2 insensitive and associated with disordered breathing in humans.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Warwick, Coventry, United Kingdom.

ABSTRACT
AbstractMutations in connexin26 (Cx26) underlie a range of serious human pathologies. Previously we have shown that Cx26 hemichannels are directly opened by CO2 (Meigh et al., 2013). However the effects of human disease-causing mutations on the CO2 sensitivity of Cx26 are entirely unknown. Here, we report the first connection between the CO2 sensitivity of Cx26 and human pathology, by demonstrating that Cx26 hemichannels with the mutation A88V, linked to Keratitis-Ichthyosis-Deafness syndrome, are both CO2 insensitive and associated with disordered breathing in humans.

Show MeSH
Related in: MedlinePlus