Pore Helix-S6 Interactions Are Critical in Governing Current Amplitudes of KCNQ3 K+ Channels

AbstractTwo 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

Pore Helix-S6 Interactions are Critical in Governing KCNQ3 Amplitudes

Idag så anser många att vi har ett samhälle där mannen har vissa privilegier kvinnan inte har och att detta är orättvist, andra menar att det är ganska jämställt och att skillnaderna mellan könen inte är så stora. Jämställdhet är ett begrepp som uppkommer i många diskussioner och används flitigt i olika styrdokument för att skapa en bra miljö för alla människor. Intresset för debatter gällande kvinnors rättigheter och villkor har ökat de senaste åren. I bland annat politiska debatter kan vi höra om exempel som att kvinnor är underbetalda inom sina yrken. Därför har det väckt vårt intresse till att undersöka hur killar som går i gymnasiet talar om jämställdhet, feminism och normer. Vi vill ta reda på hur dagens gymnasiekillar resonerar angående dessa ämnen eftersom dessa killar är och kommer att vara en del av vårt samhälle i framtiden och deras syn på jämställdhet, feminism och normer kommer att påverka utvecklingen till ett mer jämställt samhälle. Vi använder oss av gruppintervjuer där vi intervjuar fyra olika grupper med fyra personer i varje grupp. Under studiens gång så identifierar vi återkommande mönster i materialet. Dessa teman analyserar och diskuterar vi för att få en djupare och bredare förståelse av hur killar i gymnasieåldern diskuterar kring jämställdhet samt kopplingen mellan jämställdhet och feminism. Förutom feminism kopplat till jämställdhet går vi även in på vad för sorts påverkan normer i samhället har samt vilka roll(er) könen förväntas inta, enligt informanterna. De teoretiska begrepp vi använder oss av är norm, jämställdhet, och feminism

Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels

TREK/TRAAK channels are polymodal K+ channels that convert very diverse stimuli, including bioactive lipids, mechanical stretch and temperature, into electrical signals. The nature of the structural changes that regulate their activity remains an open question. Here, we show that a cytoplasmic domain (the proximal C-ter domain, pCt) exerts antagonistic effects in TREK1 and TRAAK. In basal conditions, pCt favors activity in TREK1 whereas it impairs TRAAK activity. Using the conformation-dependent binding of fluoxetine, we show that TREK1 and TRAAK conformations at rest are different, and under the influence of pCt. Finally, we show that depleting PIP2 in live cells has a more pronounced inhibitory effect on TREK1 than on TRAAK. This differential regulation of TREK1 and TRAAK is related to a previously unrecognized PIP2-binding site (R329, R330, and R331) present within TREK1 pCt, but not in TRAAK pCt. Collectively, these new data point out pCt as a major regulatory domain of these channels and suggest that the binding of PIP2 to the pCt of TREK1 results in the stabilization of the conductive conformation in basal conditions

The Role of the Carboxyl Terminus Helix C-D Linker in Regulating KCNQ3 K+ Current Amplitudes by Controlling Channel Trafficking.

In the central and peripheral nervous system, the assembly of KCNQ3 with KCNQ2 as mostly heteromers, but also homomers, underlies "M-type" currents, a slowly-activating voltage-gated K+ current that plays a dominant role in neuronal excitability. KCNQ3 homomers yield much smaller currents compared to KCNQ2 or KCNQ4 homomers and KCNQ2/3 heteromers. This smaller current has been suggested to result either from divergent channel surface expression or from a pore that is more unstable in KCNQ3. Channel surface expression has been shown to be governed by the distal part of the C-terminus in which helices C and D are critical for channel trafficking and assembly. A sequence alignment of this region in KCNQ channels shows that KCNQ3 possesses a longer linker between helix C and D compared to the other KCNQ subunits. Here, we investigate the role of the extra residues of this linker on KCNQ channel expression. Deletion of these residues increased KCNQ3 current amplitudes. Total internal reflection fluorescence imaging and plasma membrane protein assays suggest that the increase in current is due to a higher surface expression of the channels. Conversely, introduction of the extra residues into the linker between helices C and D of KCNQ4 reduced current amplitudes by decreasing the number of KCNQ4 channels at the plasma membrane. Confocal imaging suggests a higher fraction of channels, which possess the extra residues of helix C-D linker, were retained within the endoplasmic reticulum. Such retention does not appear to lead to protein accumulation and activation of the unfolded protein response that regulates protein folding and maintains endoplasmic reticulum homeostasis. Taken together, we conclude that extra helix C-D linker residues play a role in KCNQ3 current amplitudes by controlling the exit of the channel from the endoplasmic reticulum

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) regulates KCNQ3 K + channels by interacting with four cytoplasmic channel domains

Phosphatidylinositol 4,5-bisphosphate (PIP ) in the plasma membrane regulates the function of many ion channels, including M-type (potassium voltage-gated channel subfamily Q member (KCNQ), K 7) K channels; however, the molecular mechanisms involved remain unclear. To this end, we here focused on the KCNQ3 subtype that has the highest apparent affinity for PIP and performed extensive mutagenesis in regions suggested to be involved in PIP interactions among the KCNQ family. Using perforated patch-clamp recordings of heterologously transfected tissue culture cells, total internal reflection fluorescence microscopy, and the zebrafish ( ) voltage-sensitive phosphatase to deplete PIP as a probe, we found that PIP regulates KCNQ3 channels through four different domains: 1) the A-B helix linker that we previously identified as important for both KCNQ2 and KCNQ3, 2) the junction between S6 and the A helix, 3) the S2-S3 linker, and 4) the S4-S5 linker. We also found that the apparent strength of PIP interactions within any of these domains was not coupled to the voltage dependence of channel activation. Extensive homology modeling and docking simulations with the WT or mutant KCNQ3 channels and PIP were consistent with the experimental data. Our results indicate that PIP modulates KCNQ3 channel function by interacting synergistically with a minimum of four cytoplasmic domains

Opposite effects of the S4-S5 linker and PIP2 on voltage-gated channel function: KCNQ1/KCNE1 and other channels

Voltage-gated potassium (Kv) channels are tetramers, each subunit presenting six transmembrane segments (S1-S6), with each S1-S4 segments forming a voltage-sensing domain (VSD) and the four S5-S6 forming both the conduction pathway and its gate. S4 segments control the opening of the intracellular activation gate in response to changes in membrane potential. Crystal structures of several voltage-gated ion channels in combination with biophysical and mutagenesis studies highlighted the critical role of the S4-S5 linker (S4S5L) and of the S6 C-terminal part (S6T) in the coupling between the VSD and the activation gate. Several mechanisms have been proposed to describe the coupling at a molecular scale. This review summarizes the mechanisms suggested for various voltage-gated ion channels, including a mechanism that we described for KCNQ1, in which S4S5L is acting like a ligand binding to S6T to stabilize the channel in a closed state. As discussed in this review, this mechanism may explain the reverse response to depolarization in HCN-like channels. As opposed to S4S5L, the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), stabilizes KCNQ1 channel in an open state. Many other ion channels (not only voltage-gated) require PIP2 to function properly, confirming its crucial importance as an ion channel co-factor. This is highlighted in cases in which an altered regulation of ion channels by PIP2 leads to channelopathies, as observed for KCNQ1. This review summarizes the state of the art on the two regulatory mechanisms that are critical for KCNQ1 and other voltage-gated channels function (PIP2 and S4-S5L), and assesses their potential physiological and pathophysiological roles

Protein retention does not trigger the unfolded protein response (UPR).

<p>CHO cells transiently transfected with the indicated channels were lysed after 48 hours and protein loaded onto SDS-polyacrylamide gels. After transfer, PVDF membranes were probed for molecular chaperone GRP78, NF-κB and for the ER-resident protein, calreticulin (A) and UPR-related proteins such as phospho-eIF2α (B) during the UPR. As controls, CHO cells were treated with 1 ug/ml tunicamycin to induce ER stress. GAPDH and total eIF2α were used as loading controls. Shown are representative immunoblots for the indicated channels (n = 3).</p

The extra 16 helix C-D linker residues lead to channel retention in the ER.

<p>(A) Shown are representative confocal images of CHO cells expressing the indicated YFP-tagged KCNQ channels and an ER marker, pDsRED2-ER. Bars show averaged Manders coefficient for each channel which determines the degree of colocalization between the indicated channel and the ER marker (n = 19–36; **p<0.01, ***p<0.001). (B) Shown are representative confocal images of CHO cells expressing an ER marker, pDsRED2-ER with GFP-tagged 16 additional helix C-D linker residues, EGFP alone or EGFP-F (membrane-localized) (n = 11–15).</p