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Pore helix-S6 interactions are critical in governing current amplitudes of KCNQ3 K+ channels.

ABSTRACT: Two mechanisms have been postulated to underlie KCNQ3 homomeric current amplitudes, which are small compared with those of KCNQ4 homomers and KCNQ2/Q3 heteromers. The first involves differential channel expression governed by the D-helix within the C-terminus. The second suggests similar channel surface expression but an intrinsically unstable KCNQ3 pore. Here, we find H2O2-enhanced oligomerization of KCNQ4 subunits, as reported by nondenaturing polyacrylamide gel electrophoresis, at C643 at the end of the D-helix, where KCNQ3 possesses a histidine. However, H2O2-mediated enhancement of KCNQ4 currents was identical in the C643A mutant, and KCNQ3 H646C produced homomeric or heteromeric (with KCNQ2) currents similar to those of wild-type KCNQ3, ruling out this divergent residue as underlying the small KCNQ3 amplitudes. In KcsA, F103 in S6 is critical for pore-mediated destabilization of the conductive pathway. We found that mutations at the analogous F344 in KCNQ3 dramatically decreased the KCNQ3 currents. Total internal reflection fluorescence imaging revealed only minor differential surface expression among the wild-type and mutant channels. Homology modeling suggests that the effects of the F344 mutants arise from the disruption of the interaction between F344 and A315 in the pore helix. These data support a secondary role of the C-terminus, compared with pore helix-S6 interactions, in governing KCNQ3 current amplitudes.

SUBMITTER: Choveau FS

PROVIDER: S-EPMC3368123 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Pore helix-S6 interactions are critical in governing current amplitudes of KCNQ3 K+ channels.

Choveau Frank S FS Bierbower Sonya M SM Shapiro Mark S MS

Biophysical journal 20120605 11

Two mechanisms have been postulated to underlie KCNQ3 homomeric current amplitudes, which are small compared with those of KCNQ4 homomers and KCNQ2/Q3 heteromers. The first involves differential channel expression governed by the D-helix within the C-terminus. The second suggests similar channel surface expression but an intrinsically unstable KCNQ3 pore. Here, we find H2O2-enhanced oligomerization of KCNQ4 subunits, as reported by nondenaturing polyacrylamide gel electrophoresis, at C643 at the ...[more]

PMID: 22713565

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Project description: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.

Project description:The recently determined crystal structure of a chimeric Kv1.2-Kv2.1 Kv channel at 2.4 A resolution motivated this molecular-dynamics simulation study of the chimeric channel and its mutants embedded in a DPPC membrane. For the channel protein, we used two types of C-terminus: E+ and Eo. E+ contains, and Eo lacks, the EGEE residue quartet located distal to the S6 helix. For both E+ and Eo, the following trend was observed: When S4 helices were restrained at the same position as in the x-ray structure (S4high), the S6 gate remained open for 12 ns. The results were similar when the S4 helices were pulled downward 7 A (S4low). However, S4middle (or S4low) facilitated the S6 gate-narrowing for the following mutated channels (shown in order of increasing effect): 1), E395W; 2), E395W-F401A-F402A; and 3), E395W-F401A-F402A-V478W. The amino acid numbering system is that used for the Shaker channel. Even though all four subunits were set at S4low, S6 gate-narrowing was often brought about by movements of only two opposing S6 helices toward the central axis of the pore, resulting in a twofold symmetry-like structure. A free-energy profile analysis over the ion conduction pathway shows that the two opposing S6 helices whose peptide backbones are approximately 10.4 A distant from each other lead to an energetic barrier of approximately 25 kJ/mol. S6 movement was coupled with translocation of the S4-S5 linker toward the central axis of the same subunit, and the coupling was mediated by salt bridges formed between the inner (intracellular side) end of S4 and that of S6. Simulations in which S4 of only one subunit was pulled down to S4low showed that a weak intersubunit coordination is present for S5 movement, whereas the coupling between the S4-S5 linker and S6 is largely an intrasubunit one. In general, whereas subunit-based behavior appears to be dominant and to permit heteromeric conformations of the pore domain, direct intersubunit coupling of S5 or S6 is weak. Therefore, the "concerted transition" of the pore domain that has been predicted based on electrophysiological analyses is likely to be mediated mainly by the dual effects of S4 and the S4-S5 linker; these segments of one subunit can interact with both S5 of the same subunit and that of the adjacent subunit.

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Pore helix-S6 interactions are critical in governing current amplitudes of KCNQ3 K+ channels.

Publications

Pore helix-S6 interactions are critical in governing current amplitudes of KCNQ3 K+ channels.

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