Effect of charge substitutions at residue his-142 on voltage gating of connexin43 channels.
Previous studies indicate that the carboxyl terminal of connexin43 (Cx43CT) is involved in fast transjunctional voltage gating. Separate studies support the notion of an intramolecular association between Cx43CT and a region of the cytoplasmic loop (amino acids 119-144; referred to as "L2"). Structural analysis of L2 shows two alpha-helical domains, each with a histidine residue in its sequence (H126 and H142). Here, we determined the effect of H142 replacement by lysine, alanine, and glutamate on the voltage gating of Cx43 channels. Mutation H142E led to a significant reduction in the frequency of occurrence of the residual state and a prolongation of dwell open time. Macroscopically, there was a large reduction in the fast component of voltage gating. These results resembled those observed for a mutant lacking the carboxyl terminal (CT) domain. NMR experiments showed that mutation H142E significantly decreased the Cx43CT-L2 interaction and disrupted the secondary structure of L2. Overall, our data support the hypothesis that fast voltage gating involves an intramolecular particle-receptor interaction between CT and L2. Some of the structural constrains of fast voltage gating may be shared with those involved in the chemical gating of Cx43.
Shibayama
Junko
J
Department of Pharmacology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA.
Gutiérrez
Cristina
C
González
Daniel
D
Kieken
Fabien
F
Seki
Akiko
A
Carrión
Jesus Requena
JR
Sorgen
Paul L
PL
Taffet
Steven M
SM
Barrio
Luis C
LC
Delmar
Mario
M
D000595:Amino Acid Sequence; D000818:Animals; D001703:Biophysics / Q000379:methods; D018031:Connexin 43 / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018408:Patch-Clamp Techniques; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D014982:Xenopus laevis
D018031:Connexin 43; D011994:Recombinant Proteins; D006639:Histidine