Amino terminal glutamate residues confer spermine sensitivity and affect voltage gating and channel conductance of rat connexin40 gap junctions.
Connexin40 (Cx40) contains a specific binding site for spermine (affinity approximately 100 microm) whereas connexin43 (Cx43) is unaffected by identical concentrations of intracellular spermine. Replacement of two unique glutamate residues, E9 and E13, from the cytoplasmic amino terminal domain of Cx40 with the corresponding lysine residues from Cx43 eliminated the block by 2 mm spermine, reduced the transjunctional voltage (V(j)) gating sensitivity, and reduced the unitary conductance of this Cx40E9,13K gap junction channel protein. The single point mutations, Cx40E9K and Cx40E13K, predominantly affected the residual conductance state (G(min)) and V(j) gating properties, respectively. Heterotypic pairing of Cx40E9,13K with wild-type Cx40 in murine neuro2A (N2A) cells produced a strongly rectifying gap junction reminiscent of the inward rectification properties of the Kir (e.g. Kir2.x) family of potassium channels. The reciprocal Cx43K9,13E mutant protein exhibited reduced V(j) sensitivity, but displayed much less rectification in heterotypic pairings with wtCx43, negligible changes in the unitary channel conductance, and remained insensitive to spermine block. These data indicate that the connexin40 amino terminus may form a critical cytoplasmic pore-forming domain that serves as the receptor for V(j)-dependent closure and block by intracellular polyamines. Functional reciprocity between Cx40 and Cx43 gap junctions involves other amino acid residues in addition to the E or K 9 and 13 loci located on the amino terminal domain of these two connexins.
Musa
Hassan
H
Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
Fenn
Edward
E
Crye
Mark
M
Gemel
Joanna
J
Beyer
Eric C
EC
Veenstra
Richard D
RD
D000465:Algorithms; D000818:Animals; D015153:Blotting, Western; D018031:Connexin 43 / Q000502:physiology; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D017629:Gap Junctions / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000502:physiology*; D015640:Ion Channel Gating / Q000187:drug effects* / Q000235:genetics; D008239:Lysine / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D009154:Mutation; D018408:Patch-Clamp Techniques; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology*; D000097003:Gap Junction alpha-5 Protein
D018031:Connexin 43; D017630:Connexins; D018076:DNA, Complementary; D018698:Glutamic Acid; D008239:Lysine; D000644:Quaternary Ammonium Compounds; D013096:Spermine; D000097003:Gap Junction alpha-5 Protein; C027435:tetrapentylammonium