Premium
Modification of a conserved cysteine residue within S6 alters the calcium activation of hIK1 and rSK3
Author(s) -
Bailey Mark,
Devor Daniel
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a546-c
Subject(s) - gating , cysteine , chemistry , transmembrane domain , calmodulin , biophysics , transmembrane protein , mutagenesis , biochemistry , stereochemistry , amino acid , biology , mutation , enzyme , receptor , gene
Previous studies elucidating the gating mechanism for hIK1 and rSK3 have determined that calcium activation is conferred via the calcium‐calmodulin binding domain (Ca 2+ ‐CaMBD) located in the cytoplasmic C‐terminal region of these channels. We asked whether probing the channel with thimerosal (TMS), a sulfhydryl modifying reagent, would alter channel gating. The application of thimerosal to excised, inside‐out patches resulted in a Ca 2+ ‐dependent increase in channel current for hIK1, and a Ca 2+ ‐dependent decrease in channel current for rSK3. A comparison of channel sequences between hIK1 and rSK3 revealed 4 cysteine residues lining S6 of hIK1 and 2 cysteine residues lining S6 of rSK3. Site‐directed mutagenesis studies have determined that a conserved cysteine residue along the distal portion of S6 confers TMS‐sensitivity in both hIK1 and rSK3. Based on previous studies examining the inner vestibule architecture of hIK1 and rSK3, it was determined that the side‐chain of this conserved cysteine residue faces away from the ion‐conduction pathway. These results suggest that the TMS‐dependent modification of channel current may occur by disrupting the S5/S6 interaction in these channels. Furthermore, the difference in response between hIK1 and rSK3 to TMS may indicate that the interface between transmembrane domains 5 and 6 may not be conserved between these channels. These results indicate that the conformational changes necessary for mediating the coupling between Ca 2+ ‐binding and channel gating are dependent upon transmembrane interactions, particularly S5/S6. Therefore, we propose that the S5/S6 interaction is critical for coupling of Ca 2+ ‐binding to channel gating and this interaction may be different between hIK1 and rSK3.