Reconstituted connexin

Peracchia C, Shen L Gap junction channel reconstitution in artificial bilayers and evidence for calmodulin binding sites in MIP26 and connexins from rat heart, liver and xenopus embryo in Hall JE, Zampighi GA, Davis RM (eds) Gap Junctions. Progress in Cell Research, vol 3. Amsterdam, Elsevier, 1993, pp 163-170. 

D.Y. Kim, Y. Kam, S.K. Koo, C.O. Joe, Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation, J Biol Chem 274, 5581-5587 (1999). 

This chapter reviews some of the molecular biophysical questions that are raised by the properties of one class of channel-forming proteins, con-nexin, and that may be addressed through the study of connexin in reconstituted membrane systems. The first section introduces issues of biophysical interest and provides background information about connexin. The second section discusses the prospects for utilizing reconstituted systems to study the key questions, followed by a brief review of data from our laboratory. The final sections evaluate the findings and discuss future studies. 

A well-defined accessible system is required. Reconstitution into liposomes and planar phospholipid bilayers can provide the accessibility and has been used to great advantage for many ion channels (86). The disadvantages of reconstitution are that the protein may be damaged and that it may lack chemical factors required for physiological function. In addition, several unique problems arise for reconstitution of connexin channels (87). 

Development of a Reconstitution System for Study of Channels Formed by Connexin-32... 

The studies outlined in this section describe the ways we have addressed the foregoing problems of connexin reconstitution by utilizing connexin-32, the predominant form of connexin in rat liver. Our goals were to establish unambiguously that connexin-32 formed channels in liposome membranes, to identify connexin channels in planar bilayers, and to study their properties. Two methods were used to identify reconstituted channels formed by connexin-32. In one method, protein was solubilized from preparations of junctional membrane and incorporated into unilamellar liposomes. Connexin-32 was identified as a channel-forming protein by its specific enrichment in liposomes that were permeable to sucrose. In the other method, connexin-32 was affinity-purified (with a monoclonal antibody directed specifically against connexin-32) directly from octylglucoside-solubilized plasma membranes. Liposomes formed with such material were permeable to sucrose and Lucifer Yellow. Sucrose-permeable liposomes from each method were fused with planar bilayers to study the properties of connexin channels. 

Taken together, the two preceding liposome studies provide a robust demonstration that connexin-32 can be successfully reconstituted into unilamellar phospholipid membranes, where it forms pores with permeabilty similar to that of junctional channels. The data are consistent with the conducting unit being the hemichannel. 

The fundamental problems regarding reconstitution of connexin channels have been overcome connexin forms channels in unilamellar liposomes and planar bilayers. Size, permeability, and gating behavior are consistent with conducting units that are single hemichannels—the structures that span a single cell membrane and form one-half of the junctional channel. Connexin can be obtained by affinity purification under nondenaturing conditions. Thus, channels formed by a single connexin can be studied in a well-defined and accessible system. 

Presumably other connexins can be similarly purified and studied in bilayers to provide a solid basis of data on the relation between specific amino acid sequences and single-channel physiology. Variants of the connexins produced by the techniques of molecular biology can also be reconstituted, and careful comparison of the channel behavior in bilayers with that seen in cellular expression systems will be fruitful. 

It is hoped that such studies of single hemichannels will be complemented by studies of reconstituted junctional channels the double-membrane form. Development of a stable, well-characterized, and well-controlled double-membrane system is a challenging prospect. The literature on osmotic control of fusion of apposed bulged bilayers may be helpful in this regard (120, 121). Such a system would permit exploration of the forces involved in the assembly of junctional channels, which would be of interest from biophysical and cellular perspectives. For example, does the space between two membranes need to be dehydrated for hemichannels to interact (122) Do the hemichannels find each other by random interactions or does dielectric attraction (123-125) play a role Once junctional channels are formed, how reversible is the interaction between them and what forces tend to stabilize it Most important, how are the permeability, gating, and modulation of single hemichannels altered by interactions with each other in the doublemembrane form These and other considerations make the exploration of connexin channels in reconstituted systems of profound interest and promising prospects. 

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