Immunolocalization and expression of functional and nonfunctional cell-to-cell channels from wild-type and mutant rat heart connexin43 cDNA.

Abstract

The carboxyl terminal cytoplasmic domain of distinct gap junction proteins may play an important role in assembly of functional channels as well as differential responsiveness to pH, voltage, and intracellular second messengers. Oligonucleotide-directed site-specific mutagenesis in a paired Xenopus laevis oocyte expression system was used to examine the expression of mRNAs encoding wild-type and carboxyl terminal mutant connexin43 (Cx43) proteins. Oocytes were stripped, injected with mRNA or distilled water (dH2O), preincubated for 16-20 hours, and then paired for 5-10 hours; this process was followed by electrophysiological recording using the dual voltage-clamp technique. Initial experiments compared the relative junctional conductances (Gjs) in oocyte pairs expressing Cx43 (382 amino acid residues) and two truncated mutants lacking most or a portion of the cytoplasmic carboxyl terminal. The shortest mutant (M241) contained 240 amino acid residues and was devoid of all phosphorylatable serine residues in the cytoplasmic tail; its length approximated the length of liver connexin26. The longest mutant (M257) tested contained 256 amino acid residues, including two serine residues. Oocyte pairs expressing M241 yielded a Gj similar to that of oocytes injected with dH2O, whereas M257 yielded a Gj similar to that of oocytes injected with Cx43. Immunoprecipitation studies showed that Cx43, M257, and M241 proteins were readily detectable in oocytes injected with their respective mRNAs, indicating that the lack of Gj observed with the M241 mRNA was not due to reduced translation. Immunocytochemical studies revealed that wild-type and both truncated mutants were localized to the area of cell-to-cell contact between the paired oocytes, indicating that protein targeting to the membrane was not inhibited in oocytes injected with M241 mRNA. Oocyte pairs expressing mutants in which serine residues were replaced with nonphosphorylatable amino acids (serine codon No. 255 AGC was converted to GCC, alanine, designated as M255S----A, and serine codon No. 244 AGC was converted to GGC, glycine, designated as M244S----G) showed Gjs similar to M257, indicating that these serine residues and, by inference, their phosphorylation state are not critical for expression of functional channels. The importance of the length of the carboxyl terminus was assessed by comparing the Gjs in a series of mutants that were intermediate in length between M257 and M241. Gradual shortening of the carboxyl terminus produced a gradual reduction of Gj relative to M257. However, simple deletion of amino acid residues 241-257 from the wild-type Cx43 did not affect Gj relative to M257.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural Changes in the Carboxyl Terminus of the Gap Junction Protein Connexin43 Indicates Signaling between Binding Domains for c-Src and Zonula Occludens-1

“…A Cx43-ZO-1 interaction has been identified at the cardiac intercalated disc and at gap junctions in testis, Rat-1 fibroblasts, lung epithelial cells, and astrocytes (23,28). ZO-1 binding is not essential for the formation of functional Cx43 channels, as evidenced by channel formation by Cx43 constructs lacking the carboxyl terminus (54,55); however, data suggest a possible involvement of ZO-1 in the localization of Cx43 to gap junction plaques (24) as well as in the process of internalization and remodeling of Cx43 upon changes in the intracellular environment (28,56). Another possible role for the Cx43CT-ZO-1 interaction is for ZO-1 to keep in close contact cellular partners that are important in the normal function of Cx43 gap junction channels.…”

Connexin 43 Hemichannels Are Permeable to ATP

“…Truncation mutant M257 was a gift from Mario Delmar (State University of New York Upstate Medical University, Syracuse, NY). Deletion of 125 aa from the C terminal was achieved by replacing serine codon 257, TCA, with a stop codon, TGA (Dunham et al, 1992;Homma et al, 1998). Cx43-eGFP, Cx43 fused at its C terminus with enhanced green fluorescent protein (eGFP), was obtained from James Weiss (Cardiovascular Research Laboratory, University of California, Los Angeles, Los Angeles, CA) (John et al, 1999).…”

Molecular reorganization of Cx43, Zo-1 and Src complexes during the endocytosis of gap junction plaques in response to a non-genomic carcinogen

“…Although, evidence has been provided that ZO-1 is required for localization of Cx43 into gap junction plaques (Toyofuku et al, 1998), its presence is not essential for the formation of functional channels, because Cx43 constructs that lack the C-terminal region are able to form gap junction channels and plaques (Fishman et al, 1991;Dunham et al, 1992;Unger et al, 1999). Additional studies have shown that tagged Cxs are able to form gap junction plaques…”