Premium
Role of S1‐S4 salt bridges in maintaining protein stability of KCa2.3 and KCa3.1
Author(s) -
Gao Yajuan,
Chotoo Cavita K.,
Balut Corina,
Bailey Mark A.,
Devor Daniel C.
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.21.6.a955-b
Subject(s) - chemistry , transmembrane domain , gating , mutation , alanine , transmembrane protein , biophysics , amino acid , protein domain , biochemistry , gene , biology , receptor
Salt bridges in the S1‐S4 domain of voltage‐gated K + channels (Kv) are critical for maintaining protein stability and gating. Both intermediate (KCa3.1) and small (KCa2.x) conductance, calcium‐activated K + channels conserve two arginines in the S4 transmembrane domain, although they exhibit no voltage dependence in their gating. Similarly, glutamic acid and lysine residues are conserved in the S3 and S2 transmembrane domains, respectively. Thus, we determined the role of each of these amino acids in KCa3.1 and KCa2.3 by mutating them individually to alanine, glutamine or by charge‐reversal mutations (E/R or R/E). Wild type KCa3.1 has a protein half‐life of approximately 5 hours and this was dramatically decreased to between 15 and 50 min for mutations at K62 (S2), E111 (S3), R159 (S4) and R165 (S4). Similarly, we determined KCa2.3 to have a half‐life in excess of 72 hours and this was decreased to between 15 and 30 min by mutations at K321 (S2), E370 (S3), R418 (S4) and R424 (S4). A single mutation in KCa2.2 (R269A in S4) also decreased protein half‐life from approximately 16 hours to 30 min, suggesting this is a common feature across the gene family. In each case, protein degradation was prevented by lactacystin, indicating these channels were subject to proteosomal degradation. Our results suggest that salt bridges are an evolutionarily conserved means of maintaining proper structure of the S1‐S4 domain and that this feature is separate from voltage dependence.