Connexons are a large family of more than 20 members, which are distributed on all cells of the human body44. They play an important role in regulating metabolism, internal environment stability, proliferation and differentiation of cells32. Therefore, the study of connexons is of great clinical significance. The exact mechanism, by which mutant Cx26 leads to the loss of hair cells, is not clear, but it is likely to be related to the signaling molecules exchange between cells and the interaction of cochlear hair cells with the surrounding environment45. Different environmental conditions such as temperature, ion concentration, pH or voltage will affect the opening and closing functions of connexons.
Previous studies aimed to analyze such influencing factors on wild-type connexons, but we are not yet aware of the sensitivity of the mutant type of connexon to these influencing factors46,47. In the present study, we used a microarray-based technique to detect the function of wild-type connexons and mutant connexons at the cellular and microsome levels.
The temperature-dependent opening of the hemichannel human-Cx26 (hCx26) was first studied by two electrode voltage-clamp (TEVC) measurements under controlled temperature after heterologous expression of the hCx26 gene in Xenopus laevis oocytes as shown before47. The
temperature effect on the deactivating currents gives an opposite temperature dependency.
In this study, we used a novel microarray-based technique, which allows the simultaneous monitoring of different mutations on dye transport mediated by Cx26. Lucifer Yellow (LY) is a fluorescent dye, which can freely pass gap junctions and connexons48, so that we can measure the permeability of Hela cells and microsomes expressing wild-type or mutant connexons by detecting connexon opening and closing with LY.
The microsomes are spherical particles formed by broken membrane organelles from cells after mechanical homogenization. Isolation of microsomes from the nucleus, mitochondria, peroxisome and other organelles by differential centrifugation can not only retain the structural characteristics of the membrane but also keep the biological activity and function similar to living cells49. Hela cells and microsomes expressing wild-type Cx26 and the mutated Cx26 were spotted on a chip, and the uptake of LY was observed by fluorescence.
In our experiments, first we observed the uptake of LY by Hela cells expressing wild-type Cx26 under different temperature conditions by detecting the LY fluorescence signal intensity on the chip. The chips signal at cold temperature (4 degree) was much weaker than at warm
temperature (37 degree). This result is similar to a previous study demonstrating the temperature sensitivity of wild-type Cx2647.
We performed the same experiment again on Hela cells expressing mutant Cx26 (Cx26 R184P, Cx26 L90P, Cx26 F161S). L90P with the mutant of T to C transition at nucleotide 269 of the coding sequence changes a leucine at codon 90 of the second transmembrane domain to a proline is associated with non-syndromic deafness50. R184P with G to C transition at nucleotide 551 of the coding sequence changes a arginine at codon 184 of the second extracellular loop to a proline is also associated with non-syndromic deafness51.F161S lies in the second extracellular loop with a change phenylamine to serine52. All three mutations of Cx26 mentioned above lead to hereditary hearing loss. Under warm temperature conditions, the Hela cells with the mutant Cx26 showed weaker fluorescence signals than the Hela cells with the wild-type Cx26.
This result demonstrated that Hela cells expressing the mutant Cx26 uptake less LY than Hela cells expressing wild-type Cx26. Similar results were observed on microsomes expressing wild-type Cx26 and mutant Cx26. Moreover, similar results were observed on the level of the purified protein. The same experiment has been repeated on liposomes reconstituted by purified hemichannel (Cx26K188N) that were expressed in E. coli with amino acid exchange in K188 to N188.
Our experiments confirmed that the LY signal of Hela cells and microsomes expressing Cx26 with mutations was weakened. Several mutations of Cx26 can affect the channel opening and thereby limiting the ability to transmit information between neighboring cells or between the cells and its environment. With the herein presented and investigated microarray, the state of the channel opening and closure under various conditions was visible. The experimental results showed that the opening and closing function of connexons differed depending on the type of mutations. In general, different types of Cx26 mutations resulted in different structures or different hemichannel pore size: Some mutant hemichannels are completely closed and others preserving part of the connexon function.
Under 4°C cold condition, there was almost no LY uptake by Hela cells and microsomes expressing wild-type Cx26. Therefore, the LY fluorescence signals were both extremely weak. This is corroborated by the results obtained from previous research47. The same experiment was performed on Hela cells expressing mutant Cx26 and the result showed that the opening function was almost lost where connexons were in a closed state. The microsome experiment also reached the same conclusion as the cell experiment.
In low temperature environments, all types of Cx26 are basically unable to take up LY and the hemichannel is closed. As the temperature increases, the wild-type Cx26 half-channel is open. The mutant Cx26 is partially open, but the open function is decreased when compared to wild-type Cx26.
These results are similar with previous results that Cx26 hemichannels are inactive at a non-physiological temperature below 23°C47. However, this is not observed for Cx4653. How other connexin hemichannels react under temperature changes has not been investigated yet. Mutations in Cx26 influence the hemichannel activity drastically.
Carbenoxolone (CBX) is a known gap junction blocker that inhibits the function of gap junction and the opening of connexons. CBX intercalates into the plasma membrane and binds to connexons and therefore induces a conformational change which results in closure of the channel54. The effects of different concentrations of CBX on wild-type Cx26 and mutant Cx26 were examined by microarray experiments. The experimental results showed that wild-type Cx26 exhibited a dose-dependent sensitivity to CBX. We tested wild-type Cx26 and three different mutant types (Cx26 R184P, Cx26 L90P, Cx26 F161S). Mutant Cx26 R184P and L90P were less sensitive to CBX than wild-type Cx26. For F161S, we
could not find a dose-dependent effect of CBX. In this regard, microsome and cell experiments showed the same trend.
High concentrations of external free Ca2+ can inhibit the hemichannel function47 and were also used in our experimental setting to investigate the activity of the connexons. With the increase of Ca2+ concentration, hCx26 wild-type Cx26 showed a concentration-sensitive curve for the uptake ability of LY, but this was not observed in any of the mutant Cx26.
Under normal physiological conditions, the human body can regulate the function of gap junction by extracellular free Ca2+ concentration. Thereby, the transmission of intercellular ions, metabolites, signaling molecules, etc. is regulated. Depending on the type of mutation, Cx26 can lose its sensitivity to extracellular free Ca2+ concentration. When the concentration of extracellular free Ca2+ changes, connexons cannot transmit information in time leading to various diseases31.
Previous experiments regarding connexons were based on cells47,44. In our experiments, we, for the first-time, used microsomes to test the connexon function. By yielding the same results as with the use of cells, our experiments confirmed the feasibility of microsomes for the testing of connexon function. This is an important finding, since microsomes show several advantages when compared to cells. Microsomes are more stable than cells. In addition, microsomes are easy to store and can be stored for
long time periods allowing higher flexibility in the planning and performance of screenings. Furthermore, by using microsomes as a test object, the concentration is easier to adjust.
Our experiments provide an accurate and reliable method for the investigation of the channel function of Cx26. We found differences in the channel function between the tested mutant Cx26 and wild-type Cx26.
With this experimental setting, we can screen for chemically-engineered and natural compounds that can affect connexon function in future investigations. Hopefully, with the aid of this screening system, we can identify compounds that can even restore connexin functions. This is clinically relevant since connexins, a big homologous family with similar functions, control many physiological functions and their mutations or dysfunctions can be associated with various diseases such as cancer, kidney, cardiac and skin diseases. Our screening technology can be therefore used for leveraging research on all diseases linked to connexons.
Future research in our laboratory will therefore concentrate on using the herein presented microarray screening system to identify novel compounds that can influence the function of connexins. In addition, the same technology will be also applied to develop a screening system for other relevant channels or receptors.
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7. Acknowledgement
I would like to express my sincere gratitude to all people who helped me in this research work and life during my study. Thank all of you so much.
My deepest gratitude goes first and foremost to my both supervisors Prof.
Dr. med. Athanasia Warnecke from the ENT department of Hannover Medical School and PD. Dr. Carsten Zeilinger at BMWZ in Leibniz University Hannover. Thanks so much to Athanasia for providing me the so precious opportunity to study besides you and to learn in clinic, you offered me many valuable suggestions on my research, your constant encouragement and guidance supported me facing all difficulties during my study time. Thank you very much to dear Carsten, you taught me a lot of knowledge of biochemistry and experimental technique. You always gave me important advice to lead me to the right way during my research work. I got a lot from your immense professional knowledge. You have walked me through all the stages of my research and writing thesis.
Without your consistent and illuminating instruction, my project could not have reached these results. I also must thank you for your great help in my daily living.
I would like also to thank Dr. Melanie Steffens, Dr. Jennifer Schulze and Dr. Kirsten Wissel for their contribution to this research. My sincere
thanks also goes to Dr. Frank Stahl and Dr. Johanna Walter for leading me and solving many difficulties in the practical lab work. I also thank so much PD Dr. med. Martin Durisin who helped me during my internship in the ENT clinic. I am also grateful to Dozens of people who also have helped me. Dr. Marvin Peter, Dr. Qing Yue, PhD students Sona Mohammadi-Ostad-Klayeh, Lu Fan and Master student Daniel Landsberg.
I express my sincere thanks to all of you.
Last but not the least, I wish to thank my family, my parents, my loving husband Zexu Zhang and my cute daughter Yoyo, who have been assisting, supporting, and caring for me all my life. You are the source of my life.
8. Declaration
I declare that the thesis submitted to Medical School Hannover for doctoral with the title
Microarray-based screening system to investigate the activity of Connexin 26 under different conditions and mutations
was performed in the Clinic of Otorhinolaryngology and the BMWZ, under the supervision of
Prof. Dr. med. Athanasia Warnecke and PD. Dr. Carsten Zeilinger and was carried out without any other help than listed above.
The opportunity for the present doctoral procedure has not been communicated to me commercially. In particular, I did not turn on any organization for the support or the preparation of the thesis, neither completely or partially.
So far, this work has not been submitted at any German or foreign university as a thesis. Furthermore, I assure that I have not yet acquired the requested title yet.
Part of this work has been published as follows:
Wang H, Stahl F, Scheper T, Steffens M, Warnecke A, Zeilinger C.
Microarray-based screening system identifies temperature-controlled activity of Connexin 26 that is distorted by mutations. Sci Rep. 2019 Sep 19;9(1):13543. doi: 10.1038/s41598-019-49423-3.
Hannover, _______________
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