Connexin-32 channels

However, proteins in mammalian cells manage to escape from quality control in the ER to adopt alternative or dual topology in different intracellular membrane compartments. There are an increasing number of examples of proteins that are expressed in different topological forms with different functions. For example, ductin was found in two different orientations in cellular membranes, one of which serves as the subunit of the vacuolar H -ATPase and the other serves as a component of the microsomal connexin channel of gap... 

Figure 1. Schematic drawings of connexin channels. The upper figure is an edge-on view of the junctional channel in situ that spans two plasma membranes (gray areas) across extracellular space. The middle figure shows a single hemichannel, the subunit of the junctional channel that spans each membrane. The lower figure is an end-on view of a hemichannel that shows the arrangement of six connexin monomers around a...

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). 

A second problem is the identification of a bilayer channel as a relevant connexin channel. Most channels in bilayers are identified by defining physiology (e.g., ionic selectivity, toxin effects). For the junctional channel, the only certain defining properties are permeability to large molecules and, in most cases, voltage sensitivity. Other factors that affect junctional conduc-... 

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. 

From a biological perspective, the finding of functioning hemichannels seems counterintuitive. There is an understandable bias that such large channels, if they were open, would rapidly kill cells by destruction of the selective permeability of the plasma membrane. However, the plasma membrane of macrophages and mast cells can become permeable to Lucifer Yellow [possibly through connexin channels (33)] for many minutes without lethal effect (115, 116). 

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. 

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. 

C. Picoli, V. Nouvel, F. Aubry, M. Reboul, A. Duchene, T. Jeanson, J. Thomasson, F. Mouthon, M. Charveriat, Human connexin channel specificity of classical and new gap junction inhibitors, JBiomol Screen 17 (2012) 1339-1347. 

V.K. Verselis, M. Srinivas, Connexin channel modulators and their mechanisms of action. Neuropharmacology (2013). 

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