Mutagenesis Of Charged Residues In The N-terminal α-helix Of Connexin37 Reveals An Essential Lysine Residue

Abstract

Connexins are transmembrane protein subunits that combine to form cell surface hexameric hemichannels and intercellular gap junction channels. They contain a short cytoplasmic N-terminus that has been implicated in channel gating and oligomerization. Using NMR, we previously showed that the N-terminus of human connexin37 (CX37) is α-helical between amino acids 5 and 16. The α-helical region has a hydrophilic face with three aligned negatively charged residues (E8, D12 and E16) and one positively charged residue (K9). To test their function, these charged amino acid residues were mutated individually to alanines or to other neutral amino acids. The CX37 mutants were tested for formation of gap junction plaques by fluorescence microscopy after transient transfection of HeLa cells and for formation of functional hemichannels by two-microelectrode voltage clamp after expression in single Xenopus oocytes. Each of the negatively charged amino acid alanine substitution (E8A, D12A, or E16A) or charge neutralization (E8Q, D12N, or E16Q) mutants formed gap junction plaques. These mutants all formed conducting hemichannels; ionic currents were of comparable magnitude to those in oocytes expressing wild-type CX37 when measured without divalent cations and were blocked by 2 mM external calcium. While gap junction plaques were observed in HeLa cells transfected with K9A, this construct did not form conducting hemichannels in Xenopus oocytes. No plaques were detected in a charge reversal mutant at this position (K9E). These results suggest that the negatively charged residues within the N-terminal α-helix are not individually required for formation of functional channels. In contrast, the positively charged residue, K9, is required for hemichannel opening and influences formation of gap junction plaques.