DOI QR코드

DOI QR Code

Single Channel Analysis of Xenopus Connexin 38 Hemichannel

제노푸스 Cx38 세포막채널의 단일채널분석

  • Cheon, Mi-Saek (Department of Physiology, College of Medicine, Dankook University) ;
  • Oh, Seung-Hoon (Department of Physiology, College of Medicine, Dankook University)
  • 천미색 (단국대학교 의과대학 생리학교실) ;
  • 오승훈 (단국대학교 의과대학 생리학교실)
  • Published : 2007.11.30

Abstract

Gap junction channels formed by two adjacent cells allow the passage of small molecules up to ${\sim}\;1\;kDa$ between them. Hemichannel (connexon or half of gap junction) also behaves as a membrane channel like sodium or potassium channels in a single cell membrane. Among 26 types of connexin (Cx), $Cx32^*43E1$ (a chimera in which the first extracellular loop of Cx32 has been replaced with that of Cx43), Cx38, Cx46, and Cx50 form functional hemichannels as well as gap junction channels. Although it is known that Xenopus oocytes express endogenous connexin 38 (Cx38), its biophysical characteristics at single channel level are poorly understood. In this study, we performed single channel recordings from single Xenopus oocytes to acquire the biophysical properties of Cx38 including voltage-dependent gating and permeation (conductance and selectivity). The voltage-dependent fast and slow gatings of Cx38 hemichannel are distinct. Fast gating events occur at positive potentials and their open probabilities are low. In contrast, slow gatings dominate at negative potentials with high open probabilites. Based on hi-ionic experiments, Cx38 hemichannel is anion-selective. It will be interesting to test whether charged amino acid residues in the amino terminus of Cx38 are responsible for voltage gatings and permeation.

간극결합(intercellular channel)은 인접하는 두 세포사이에 형성된 이온채널이며 이를 통해서 각종 이온, 이차 신호전달물질, 그리고 1 kDa 미만의 대사물질들이 통과한다. 아울러, sodium 혹은 potassium 이온채널처럼 반쪽의 간극결합(connexon 혹은 hemichannel)도 세포막채널로서 작용을 한다. 현재까지 간극결합을 구성하는 connexin (Cx) 단위체는 26종류 이상이 확인되었다. 이 가운데, Cx32, Cx38, Cx46 그리고 Cx50 만이 간극결합채널뿐만 아니라 세포막채널로서도 기능을 수행한다. Xenopus oocytes에서 connexin 38 (Cx38)이 발현하는 것으로 알려져 있지만 Cx38의 생물리학적 특성이 단일채널수준에서 연구가 진행된 경우는 없다. 이번 연구에서는 Cx38 채널의 생물리학적 특성, 즉 전압-의존적 개폐와 투과성(전기전도도와 이온선택성)을 알아보고자 단일채널기록을 수행하였다. Cx38 hemichannel은 전압-의존적인 빠른 개폐와 느린 개폐의 특성을 보였다. 양성전압 환경에서는 Cx38 채널이 낮은 열릴 확률(open probability)로 빠른 개폐가 유도된 반면, 음성전압에서는 느린 개폐가 높은 열릴 확률로 유도되었다. bi-ionic 실험을 통하여, Cx38 채널은 양이온보다 음이온을 더 선택 적으로 통과시킨다는 점을 알게 되었다. Cx38의 아미노산서열을 살펴보면, 아미노말단부위에 전하를 띠는 5개의 아미노산 잔기가 존재한다. 앞으로 이들 잔기를 치환시킨 돌연변이 Cx38 채널을 이용하여 과연 이들 아미노산 부위가 전압-의존적 개폐와 투과성에 관여하는 지 여부를 조사하는 연구는 매우 흥미로운 결과를 도출할 것으로 기대한다.

Keywords

References

  1. Brink, P. R. and M. M. Dewey. 1980. Evidence for fixed charge in the nexus. Nature 285, 101-202. https://doi.org/10.1038/285101a0
  2. Bukauskas, F. F., A. Bukauskiene and V. K. Verselis. 2002. Conductance and permeability of the residual state of connexin43 gap junction channels. J. Gen. Physiol. 119, 171-186. https://doi.org/10.1085/jgp.119.2.171
  3. Ebihara, L. 1996. Xenopus connexin38 forms hemi-gapjunctional channels in the nonjunctional plasma membrane of Xenopus oocytes. Biophys. J. 71, 742-748. https://doi.org/10.1016/S0006-3495(96)79273-1
  4. Ebihara, L., E. C. Beyer, K. I. Swenson, D. L. Paul and D. A. Goodenough. 1989. Cloning and expression of a Xenopus embryonic gap junction protein. Science 243, 1194-1195. https://doi.org/10.1126/science.2466337
  5. Eisenman, G. and R. Horn. 1983. Ionic selectivity revisited: the role of kinetic and equillibrium processes in ion permeation through channels. J. Membr. Biol. 76, 197-225. https://doi.org/10.1007/BF01870364
  6. Gimlich, R. L., N. M. Kumar and N. B. Gilula. 1990. Differential regulation of the levels of three gap junction mRNAs in Xenopus embryos. J. Cell Biol. 110, 597-605. https://doi.org/10.1083/jcb.110.3.597
  7. Harris, A. L. 2001. Emerging issues of connexin channels: Biophysics fills the gap. Q. Rev. Biophys. 34, 325-472.
  8. Oh, S., C. K. Abrams, V. K. Verselis and T. A. Bargiello. 2000. Stoichiometry of transjunctional voltage-gating polarity reversal by a negative charge substitution in the amino terminus of a connexin32 chimera. J. Gen. Physiol. 116, 13-31. https://doi.org/10.1085/jgp.116.1.13
  9. Oh, S., Y. Ri, M. V. Bennett, E. B. Trexler, V. K. Verselis and T. A. Bargiello. 1997. Changes in permeability caused by connexin 32 mutations underlie X-linked Charcot- Marie-Tooth disease. Neuron 19, 927-938. https://doi.org/10.1016/S0896-6273(00)80973-3
  10. Oh, S., S. Rivkin, Q. Tang, V. K. Verselis and T. A. Bargiello. 2004. Determinants of gating polarity of a connexin 32 hemichannel. Biophys. J. 87, 912-928 https://doi.org/10.1529/biophysj.103.038448
  11. Oh, S., J. B. Rubin, M. V. L. Bennett, V. K. Verselis and T. A. Bargiello. 1999. Molecular determinants of electrical rectification of single channel conductance in gap junctions formed by connexin 26 and 32. J. Gen. Physiol. 114, 339-364. https://doi.org/10.1085/jgp.114.3.339
  12. Pfahnl, A., X. W. Zhou, R. Werner and G. Dahl. 1997. A chimeric connexin forming gap junction hemichannels. Pflugers Arch. 433, 773-779. https://doi.org/10.1007/s004240050344
  13. Rubin, J. B., V. K. Verselis, M. V. Bennett and T. A. Bargiello. 1992. A domain substitution procedure and its use to analyze voltage dependence of homotypic gap junctions formed by connexins 26 and 32. Proc. Natl. Acad. Sci. USA 89, 3820-3824. https://doi.org/10.1073/pnas.89.9.3820
  14. Rubin, J. B., V. K. Verselis, M. V. Bennett and T. A. Bargiello. 1992. Molecular analysis of voltage dependence of heterotypic gap junctions formed by connexins 26 and 32. Biophys. J. 62, 183-193. https://doi.org/10.1016/S0006-3495(92)81804-0
  15. Trexler, E. B., M. V. Bennett, T. A. Bargiello and V. K. Verselis. 1996. Voltage gating and permeation in a gap junction hemichannel. Proc. Natl. Acad. Sci. USA 93, 5836-5841. https://doi.org/10.1073/pnas.93.12.5836
  16. Verselis, V. K., C. S. Ginter and T. A. Bargiello. 1994. Opposite voltage gating polarities of two closely related connexins. Nature 368, 348-351 https://doi.org/10.1038/368348a0
  17. Waltzman, M. N. and D. C. Spray. 1995. Exogenous expression of connexins for physiological characterisation of channel properties: Comparison of methods and results, pp. 9-17, In Kanno, Y., K. Kataoka, Y. Shiba, Y. Shibata and T. Shimazu (eds.), International Communication Through Gap Junctions: Progress in Cell Research, Vol. 4, Elsevier, Amsterdam.
  18. Yim, J., M. Cheon, J. Jung and S. Oh. 2006. Effect of amino terminus of gap junction hemichannel on its channel gating. J. Life Sci. 16, 37-43. https://doi.org/10.5352/JLS.2006.16.1.037