title: Role of the C-terminus of the high-conductance calcium-activated potassium channel in channel structure and function.
abstract: The role of ion channels in cell physiology is regulated by processes occurring after protein biosynthesis, which are critical for both channel function and targeting of channels to appropriate cell compartments. Here we apply biochemical and electrophysiological methods to investigate the role of the high-conductance, calcium-activated potassium (Maxi-K) channel C-terminal domain in channel tetramerization, association with the beta1 subunit, trafficking of the channel complex to the cell surface, and channel function. No evidence for channel tetramerization, cell surface expression, or function was observed with Maxi-K(1)(-)(323), a construct truncated three residues after the S(6) transmembrane domain. However, Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) are able to form tetramers and to associate with the beta1 subunit. Maxi-K(1)(-)(343)-beta1 and Maxi-K(1)(-)(441)-beta1 complexes are efficiently targeted to the cell surface and cannot be pharmacologically distinguished from full-length channels in binding experiments but do not form functional channels. Maxi-K(1)(-)(651) forms tetramers and associates with beta1; however, the complex is not present at the cell surface, but is retained intracellularly. Maxi-K(1)(-)(651) surface expression and channel function can be fully rescued after coexpression with its C-terminal complement, Maxi-K(652)(-)(1113). However coexpression of Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) with their respective C-terminal complements did not rescue channel function. Together, these data demonstrate that the domain(s) in the Maxi-K channel necessary for formation of tetramers, coassembly with the beta1 subunit, and cell surface expression resides within the S(0)-S(6) linker domain of the protein, and that structural constraints within the gating ring in the C-terminal region can regulate trafficking and function of constructs truncated in this region.
authors.affiliation: Department of Ion Channels, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065, USA.
authors.forename: William A
authors.initials: WA
authors.lastname: Schmalhofer
authors.forename: Manuel
authors.initials: M
authors.lastname: Sanchez
authors.forename: Ge
authors.initials: G
authors.lastname: Dai
authors.forename: Ashvin
authors.initials: A
authors.lastname: Dewan
authors.forename: Lorena
authors.initials: L
authors.lastname: Secades
authors.forename: Markus
authors.initials: M
authors.lastname: Hanner
authors.forename: Hans-Guenther
authors.initials: HG
authors.lastname: Knaus
authors.forename: Owen B
authors.initials: OB
authors.lastname: McManus
authors.forename: Martin
authors.initials: M
authors.lastname: Kohler
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kaczorowski
authors.forename: Maria L
authors.initials: ML
authors.lastname: Garcia
mesh_terms: D001224:Aspartic Acid / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D051036:Large-Conductance Calcium-Activated Potassium Channels; D007470:Monoiodotyrosine / Q000235:genetics; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D026902:Potassium Channel Blockers / Q000378:metabolism; D024681:Potassium Channels, Calcium-Activated / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D012604:Scorpion Venoms / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000235:genetics
chemical_list: D007457:Iodine Radioisotopes; D051036:Large-Conductance Calcium-Activated Potassium Channels; D010446:Peptide Fragments; D010455:Peptides; D026902:Potassium Channel Blockers; D024681:Potassium Channels, Calcium-Activated; D021122:Protein Subunits; D012604:Scorpion Venoms; D001224:Aspartic Acid; D014443:Tyrosine; D010649:Phenylalanine; C064719:iberiotoxin; D007470:Monoiodotyrosine