Functional analysis of human cardiac gap junction channel mutants.
The connexins form a family of membrane spanning proteins that assemble into gap junction channels. The biophysical properties of these channels are dependent upon the constituent connexin isoform. To begin identifying the molecular basis for gap junction channel behavior in the human heart, a tissue that expresses connexin43, we used site-directed mutagenesis to generate mutant cDNAs of human connexin43 with shortened cytoplasmic tail domains. Premature stop codons were inserted, resulting in proteins corresponding in length to the mammalian isoforms connexin32 and connexin26, which are expressed primarily in liver. All constructs restore intercellular coupling when they are transfected into SKHep1 cells, a human hepatoma line that is communication deficient. Whereas wild-type connexin43 transfectants display two distinct unitary conductance values of about 60 and 100 pS, transfectants expressing the mutant proteins, from which 80 and 138 amino acids have been deleted, exhibit markedly different single-channel properties, with unitary conductance values of about 160 and 50 pS, respectively. Junctional conductance of channels composed of wild-type connexin43 is less voltage-sensitive compared with transfectants expressing wild-type connexin32. However, neither of the connexin43 truncation mutants alters this relative voltage insensitivity. These results suggest that the cytoplasmic tail domain is an important determinant of the unitary conductance event of gap junction channels but not their voltage dependence. Furthermore, since the mutant connexins are missing several consensus phosphorylation sites, modification of these particular sites may not be required for membrane insertion or assembly of human connexin43 into functional channels.
Fishman
G I
GI
Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461.
Moreno
A P
AP
Spray
D C
DC
Leinwand
L A
LA
D001692:Biological Transport; D015139:Blotting, Southern; D003001:Cloning, Molecular; D000072259:Connexin 26; D017630:Connexins; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D006801:Humans; D066298:In Vitro Techniques; D007365:Intercellular Junctions / Q000502:physiology*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014162:Transfection
D017630:Connexins; C000607042:GJB2 protein, human; D008565:Membrane Proteins; D012333:RNA, Messenger; D000072259:Connexin 26; D004247:DNA