Details
| Original language | English |
|---|---|
| Article number | 13543 |
| Journal | Scientific reports |
| Volume | 9 |
| Issue number | 1 |
| Early online date | 19 Sept 2019 |
| Publication status | E-pub ahead of print - 19 Sept 2019 |
Abstract
Here, we show that human Connexin 26 (hCx26 or Cx26WT) hemichannel opening rapidly enables the transport of small molecules when triggered by temperature and by compensation of the Ca2+ blockade with EDTA. Point mutations within Cx26 were analysed by a novel optical microarray-based Lucifer Yellow uptake assay or by two electrode voltage clamp (TEVC) on frog oocytes to monitor simultaneous activities of channel proteins. Point mutations L90P, F161S, R184P or K188N influenced the temperature-dependent activity drastically. Since several mutations blocked trafficking, the temperature-dependent activity of the recombinant synthesized and purified wild-type Cx26WT and Cx26K188N hemichannel was tested by liposome flux assay (LFA) and on a microarray-based Lucifer Yellow uptake assay under warm conditions (>30 °C). The data from TEVC measurements and dye flux experiments showed that the mutations gave no or only a weak activity at increased temperature (>30 °C). We conclude that the position K188 in the Cx26WT forms a temperature-sensitive salt bridge with E47 whereas the exchange to K188N destabilizes the network loop- gating filter, which was recently identified as a part of the flexible Ca2+ binding site. We assume that the temperature sensitivity of Cx26 is required to protect cells from uncontrolled release or uptake activities through Cx26 hemichannels.
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In: Scientific reports, Vol. 9, No. 1, 13543, 19.09.2019.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Microarray-based screening system identifies temperature-controlled activity of Connexin 26 that is distorted by mutations
AU - Wang, Hongling
AU - Stahl, Frank
AU - Scheper, Thomas
AU - Steffens, Melanie
AU - Warnecke, Athanasia
AU - Zeilinger, Carsten
N1 - Funding information: We are grateful to Klaus Willecke for supplying HeLa cell lines Hongling Wang was financed by Elite Young Doctors study abroad plan of Chineses People’s Armed PoDMEM Characteristic Medical Center. The publication of this article was funded by the Open Access Fund of the Leibniz Universität Hannover.
PY - 2019/9/19
Y1 - 2019/9/19
N2 - Here, we show that human Connexin 26 (hCx26 or Cx26WT) hemichannel opening rapidly enables the transport of small molecules when triggered by temperature and by compensation of the Ca2+ blockade with EDTA. Point mutations within Cx26 were analysed by a novel optical microarray-based Lucifer Yellow uptake assay or by two electrode voltage clamp (TEVC) on frog oocytes to monitor simultaneous activities of channel proteins. Point mutations L90P, F161S, R184P or K188N influenced the temperature-dependent activity drastically. Since several mutations blocked trafficking, the temperature-dependent activity of the recombinant synthesized and purified wild-type Cx26WT and Cx26K188N hemichannel was tested by liposome flux assay (LFA) and on a microarray-based Lucifer Yellow uptake assay under warm conditions (>30 °C). The data from TEVC measurements and dye flux experiments showed that the mutations gave no or only a weak activity at increased temperature (>30 °C). We conclude that the position K188 in the Cx26WT forms a temperature-sensitive salt bridge with E47 whereas the exchange to K188N destabilizes the network loop- gating filter, which was recently identified as a part of the flexible Ca2+ binding site. We assume that the temperature sensitivity of Cx26 is required to protect cells from uncontrolled release or uptake activities through Cx26 hemichannels.
AB - Here, we show that human Connexin 26 (hCx26 or Cx26WT) hemichannel opening rapidly enables the transport of small molecules when triggered by temperature and by compensation of the Ca2+ blockade with EDTA. Point mutations within Cx26 were analysed by a novel optical microarray-based Lucifer Yellow uptake assay or by two electrode voltage clamp (TEVC) on frog oocytes to monitor simultaneous activities of channel proteins. Point mutations L90P, F161S, R184P or K188N influenced the temperature-dependent activity drastically. Since several mutations blocked trafficking, the temperature-dependent activity of the recombinant synthesized and purified wild-type Cx26WT and Cx26K188N hemichannel was tested by liposome flux assay (LFA) and on a microarray-based Lucifer Yellow uptake assay under warm conditions (>30 °C). The data from TEVC measurements and dye flux experiments showed that the mutations gave no or only a weak activity at increased temperature (>30 °C). We conclude that the position K188 in the Cx26WT forms a temperature-sensitive salt bridge with E47 whereas the exchange to K188N destabilizes the network loop- gating filter, which was recently identified as a part of the flexible Ca2+ binding site. We assume that the temperature sensitivity of Cx26 is required to protect cells from uncontrolled release or uptake activities through Cx26 hemichannels.
UR - http://www.scopus.com/inward/record.url?scp=85072372797&partnerID=8YFLogxK
U2 - 10.1038/s41598-019-49423-3
DO - 10.1038/s41598-019-49423-3
M3 - Article
C2 - 31537823
AN - SCOPUS:85072372797
VL - 9
JO - Scientific reports
JF - Scientific reports
SN - 2045-2322
IS - 1
M1 - 13543
ER -