Connexins 32 and

Charcot-Marie-Tooth disease and other inherited neuropathies AD, AR or X-linked PMP-22, P0, connexin-32 and other genes Variable degrees of myelin deficiency specific for the PNS see text 1,28-30... 

Kwa MSG, van Schaik IN, Brand A, Baas F, Veimeulen M (2001) Invesdgadon of serum response to PMP22, connexin 32 and P-0 in inflammatory neuropadiies. J Neuroimmunol 116 220—225. 

The argument against sucrose permeability being due to a nonconnexin protein is as follows If the sucrose permeability was due to a hypothetical nonconnexin protein, and the proteins were distributed independently of one another in the liposomes, then connexin-32 and its fragment would be present in the same ratio in the two liposome populations, each distributed independently of sucrose permeability. Because they are not, it is reasonable to conclude that connexin-32 causes a permeability to sucrose. The presence of some fragment in the sucrose-permeable liposomes is accounted for by its presence in liposomes that also contained full-length (functional) connexin. The conclusion that connexin-32 forms a pore does not require that all nonconnexin protein be excluded from the sucrose-permeable liposomes or that connexin-32 account for all of the sucrose permeability. It relies on the positive and specific correlation between sucrose permeability and enrichment for full-length connexin-32. 

Saez JC, Naim AC, Czemick A, Spray DC, Hertzberg EL, Greengard P, Bennett MVL Phosphorylation of connexin 32, the hepatocyte gap junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca/calmoduUn-dependent protein kinase II. Eur J Biochem 1990 192 263-273. 

M., Nelles, E., Willecke, K., Zielasek, J., et al. (1997) Structural abnormalities and deficient maintenance of peripheral nerve myelin in mice lacking the gap junction protein connexin 32. J Neurosci 17, 4545-4551. 

PNS myelin proteins include two abundant constituents glycoprotein zero (PO) and MBPS (already discussed in III. 1), and a set of minor ones including PMP22, MAG, and connexin 32 (already discussed in III.l). 

Rash JE, Staines WA, Yasmura T, Patel D, Eurman DS, et al. 2000. Immunogold evidence that the neuronal gap junctions in adult rat brain and spinal cord contain connexin-36 but not connexin-32 or connexin-43. Proc Nat Acad Sci USA 97 7573-7578,... 

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. 

A significant fraction of the liposomes that contain junctional protein were sucrose-permeable (Figure 2D and E). This result shows a correlation between protein from junctional membrane and sucrose permeability, but does not, by itself, permit the conclusion that connexin-32 forms the sucrose-permeable pathway. 

To identify the protein responsible for the permeability, proteins in the liposomes were separated by standard denaturing electrophoresis, blotted, and stained for total protein and for connexin-32. Western blots of unfractionated liposomes formed in the presence of protein solubilized from isolated gap junctions are shown in Figure 3A. The blots show the monomeric, dimeric, and trimeric forms of connexin-32 commonly observed in sodium dodecyl sulfate (SDS) gels of isolated junctions (59-61, 107). They also show the presence of a commonly seen proteolytic fragment of connexin-32 (59, 61, 107) (better seen in Figure 3B), which contributes to the broadness of the staining below the monomer and dimer bands. The liposomes typically contained no detectable nonconnexin protein. 

Affinity-Purified Connexin-32 Forms a Large Pore in Liposomes (103). Connexin-32 solubilized in octylglucoside from crude plasma membrane was affinity-purified under nondenaturing conditions using a monoclonal antibody specific for connexin-32 (7) that was attached to a bead matrix (98 and Rhee, S. K. Harris, A. L., unpublished). Overloaded denatur-... 

The affinity-purified connexin-32 was incorporated into unilamellar liposomes as before. The connexin-32 induced a sucrose permeability in liposomes, as assayed by the density-shift technique, and gave results essentially identical to those in Figure 2. Liposomes that were sucrose-permeable did not retain the dye Lucifer Yellow (retained by the sucrose-impermeable liposomes), which is near the upper size-permeability limit for gap junction channels (19, 108, 109). The fraction of the liposomes that were permeable to sucrose decreased by a factor of 4 when the pH in the gradients was changed from 7.5 to 6.0. This effect was partially reversible. 

Figure 5. Voltage dependence of P0 for the larger (130-pS) connexin-32 channels. A, Fit to ln( P0/Pj) as a function of voltage. B, Fit to P0 as a function of voltage. The solid line is calculated with the Boltzmann relation (SSE — 0.007) and the dashed line is an independent fit (SSE = 0.001).

What do these liposome and bilayer data imply about connexin-32 channels ... 

Figure 7. Bilayer conductance induced by affinity-purified connexin-32. A, Sucrose-permeable liposomes formed with affinity-purified connexin-32 were fused with planar phospholipid bilayers as described. Highly filtered (5-Hz comer frequency) currents show unstable conductances, but large, rapid fluctuations that cluster around multiples of about 125 pS may be discerned (arrowheads). The bilayer voltage was 50 mV. B, Higher resolution recording of channels from affinity-purified connexin-32. Records show discrete gating conductance transitions, but with a high rate and amplitude of current fluctuations through the open channels. Unitary conductance is difficult to determine, but is near 200 pS. The bilayer voltage was 100 mV.

Kamasawa N, Sik A, Morita M et al (2005) Connexin-47 and connexin-32 in gap junctions of oligodendrocyte somata, myelin sheaths, paranodal loops and Schmidt-Lanterman indsmes implications for ionic homeostasis and potassimn siphoning. Neuroscience 136 65-86... 

Another connexin with a molecular weight of 32 kD, Cx32, was cloned from human liver [Kumar and Gilula, 1986], rat liver [Paul, 1986] and was also found in hepatocytes [Paul, 1986 Traub et ah, 1989], stomach, brain and kidney [Paul, 1986] as well as in pancreatic acinar cells [Dermietzel et ah,... 

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