N-terminal residues in Cx43 and Cx40 determine physiological properties of gap junction channels, but do not influence heteromeric assembly with each other or with Cx26.
The cytoplasmic N-terminal domain in the connexins (Cx) has been implicated in determining several properties including connexin hetero-oligomerization, channel gating and regulation by polyamines. To elucidate the roles of potentially crucial amino acids, we produced site-directed mutants of connexins Cx40 and Cx43 (Cx40E12S,E13G and Cx43D12S,K13G) in which the charged amino acids at positions 12 and 13 were replaced with serine and glycine as found in Cx32. HeLa, N2a and HEK293 cells were transfected and studied by immunochemistry and double whole-cell patch clamping. Immunoblotting confirmed production of the mutant proteins, and immuno-fluorescence localized them to punctuate distributions along appositional membranes. Cx40E12S,E13G and Cx43D12S,K13G formed homotypic gap junction channels that allowed intercellular passage of Lucifer Yellow and electrical current, but these channels exhibited negligible voltage-dependent gating properties. Unlike wild-type Cx40, Cx40E12S,E13G channels were insensitive to block by 2 mM spermine. Affinity purification of material solubilized by Triton X-100 from cells co-expressing mutant Cx43 or mutant Cx40 with wild-type Cx40, Cx43 or Cx26 showed that introducing the mutations did not affect the compatibility or incompatibility of these proteins for heteromeric mixing. Co-expression of Cx40E12S,E13G with wild-type Cx40 or Cx43 dramatically reduced voltage-dependent gating. Thus, whereas the charged amino acids at positions 12 and 13 of Cx40 or Cx43 are not required for gap junction assembly or the compatibility of oligomerization with each other or with Cx26, they strongly influence several physiological properties including those of heteromeric channels.
Gemel
Joanna
J
Department of Pediatrics, Section of Hematology/Oncology and Stem Cell Transplantation, University of Chicago, IL 60637-1470, USA.
Lin
Xianming
X
Veenstra
Richard D
RD
Beyer
Eric C
EC
D002460:Cell Line; D000072259:Connexin 26; D018031:Connexin 43 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D017630:Connexins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D017629:Gap Junctions / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D013096:Spermine / Q000494:pharmacology; D000097003:Gap Junction alpha-5 Protein
D000072259:Connexin 26; D018031:Connexin 43; D017630:Connexins; D013096:Spermine; D000097003:Gap Junction alpha-5 Protein; C000607042:GJB2 protein, human