Connexins isolation

A connexin with a molecular weight of 30 kD (Cx30) has been isolated and cloned from xenopus liver and was also found in the lung, intestine, stomach and kidney of xenopus [Gimlich et ah, 1988]. The C terminus is enlarged to 58 amino acids. The N terminus contains 22 amino acids. The amino acid sequence is also given in figure 6. 

Often the question arises, whether the total amount of connexin is altered. Because immunohistology is mostly a semiquantitative method, the following biochemical method for isolation of gap junctions may be a suitable alternative. After isolation of the gap junction pellet SDS-PAGE has to be carried out and the gels have to be stained. For control an immunoblot is recommended. 

The role of GJ in ischemia and reperfusion injury has been shown in studies using GJ blockers reduction of necrosis after ischemia and reperfusion was observed in in situ rabbit hearts and in isolated rat hearts with administration of halothane (presumed to be a GJ uncoupling agent).23,24 Furthermore, regulation of the phosphorylation of connexin 43 (non phosphorylated Cx43 increases the opening of GJ) resulted in modification of ischemic injury. In fact, ischemic preconditioning (brief episodes of ischemia and reperfusion) induced cardioprotection was associated with preservation of connexin 43 phosphorylation.25... 

L. Polontchouk, V. Valiunas, J.-A. Haefliger, H.M. Eppenberger, R. Weingart, Expression and regulation of connexins in cultured ventricular myocytes isolated from adult rat hearts, Pfliigers Arch - Eur J Physiol 443, 676-689 (2002). 

R.P. Kondo, SY. Wang, SA. John, JN. Weiss, JI. Goldhaber, Metabolic inhibition activates a non-selec-tive current through connexin hemichannels in isolated ventricular myocytes, J Mol Cell Cardiol 32, 1859-1872 (2000). 

As a prerequisite for fine motor control, skeletal muscle fibers are electrically isolated from one another, and, accordingly, do not express either connexin-43 (the major gap junction protein) or N-cadherin (the major adherens protein in cardiac intercalated discs). Asynchronous islands of intramyocardial skeletal muscle can result in lethal arrhythmias in mice [21]. Although cell types such as fetal cardiomyocytes [22] and cardiomyocytes derived from murine or human embryonic stem cells [23] are capable of electromechanical coupling, their clinical use has unfortunately been hampered by technical, ethical, moral, social and legal hurdles. 

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. 

Bilayer Channels from Sucrose-Permeable Liposomes (87, 101, 102). Sucrose-permeable liposomes from the foregoing studies were fused with planar phospholipid bilayers (110, 111). The data from the liposomes that contain connexin from isolated junctions differed in some respects from the data obtained with the affinity-purified connexin. At the present time, the data from the affinity-purified material is not fully characterized. Therefore, most of the data described in the following text are from liposomes that contain connexin-32 from isolated junctions exceptions are noted. 

The starting material for the affinity purification was predominantly plasma membrane from presumably well-coupled cells therefore, the con-nexin that is isolated is most likely from junctional structures. Because the purified connexin-32 is predominantly in structures the size of single hemichannels, a simple conclusion is that octylglucoside can disrupt the end-to-end interactions of junctional channels. However, there is the caveat that the junctions may split prior to solubilization, that is, in the preparation of the crude plasma membrane fraction. Junctions can split when a tissue is perfused with hyperosmotic solutions (II7), so a cellular splitting could precede disruption of junctional channels by octylglucoside. 

---