title: Determinants within the turret and pore-loop domains of KCNQ3 K+ channels governing functional activity.
abstract: KCNQ1-5 (Kv7.1-7.5) subunits assemble to form a variety of functional K(+) channels in the nervous system, heart, and epithelia. KCNQ1 and KCNQ4 homomers and KCNQ2/3 heteromers yield large currents, whereas KCNQ2 and KCNQ3 homomers yield small currents. Since the unitary conductance of KCNQ3 is five- to 10-fold greater than that of KCNQ4 or KCNQ1, these differences are even more striking. To test for differential membrane protein expression, we performed biotinylation and total internal reflection fluorescence imaging assays; however, both revealed only small differences among the channels, leading us to investigate other mechanisms at work. We probed the molecular determinants governing macroscopic current amplitudes, with focus on the turret and pore-loop domains of KCNQ1 and KCNQ3. Elimination of the putative N289 glycosylation site in KCNQ1 reduced current density by approximately 56%. A chimera consisting of KCNQ3 with the turret domain (TD) of KCNQ1 increased current density by about threefold. Replacement of the proximal half of the TD in KCNQ3 with that of KCNQ1 increased current density by fivefold. A triple chimera containing the TD of KCNQ1 and the carboxy terminus of KCNQ4 yielded current density 10- or sixfold larger than wild-type KCNQ3 or KCNQ1, respectively, suggesting that the effects on current amplitudes of the TD and the carboxy-terminus are additive. Critical was the role of the intracellular TEA(+)-binding site. The KCNQ3 (A315T) swap increased current density by 10-fold, and the converse KCNQ1 (T311A) swap reduced it by 10-fold. KCNQ3 (A315S) also yielded greatly increased current amplitudes, whereas currents from mutant A315V channels were very small. The KCNQ3 (A315T) mutation increased the sensitivity of the channels to external Ba(2+) block by eight- to 28-fold, consistent with this mutation altering the structure of the selectivity filter. To investigate a structural hypothesis for the effects of these mutations, we performed homology modeling of the pore region of wild-type and mutant KCNQ3 channels, using KvAP as a template. The modeling suggests a critical stabilizing interaction between the pore helix and the selectivity filter that is absent in wild-type KCNQ3 and the A315V mutant, but present in the A315T and A315S mutants. We conclude that KCNQ3 homomers are well expressed at the plasma membrane, but that most wild-type channels are functionally silent, with rearrangements of the pore-loop architecture induced by the presence of a hydroxyl-containing residue at the 315 position "unlocking" the channels into a conductive conformation.
authors.affiliation: Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA.
authors.forename: Oleg
authors.initials: O
authors.lastname: Zaika
authors.forename: Ciria C
authors.initials: CC
authors.lastname: Hernandez
authors.forename: Manjot
authors.initials: M
authors.lastname: Bal
authors.forename: Gleb P
authors.initials: GP
authors.lastname: Tolstykh
authors.forename: Mark S
authors.initials: MS
authors.lastname: Shapiro
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001464:Barium / Q000494:pharmacology; D001665:Binding Sites; D019921:Biotinylation; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004553:Electric Conductivity; D005786:Gene Expression Regulation; D006801:Humans; D051659:KCNQ3 Potassium Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D016062:Porosity; D026902:Potassium Channel Blockers / Q000494:pharmacology; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D019789:Tetraethylammonium / Q000378:metabolism
chemical_list: D051659:KCNQ3 Potassium Channel; D026902:Potassium Channel Blockers; D001464:Barium; D019789:Tetraethylammonium
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