TY - JOUR AU - Szántó, Gábor Tibor AU - Papp, Ferenc AU - Zákány, Florina AU - Varga, Zoltán AU - Deutsch, Carol AU - Panyi, György TI - Molecular rearrangements in S6 during slow inactivation in Shaker -IR potassium channels JF - JOURNAL OF GENERAL PHYSIOLOGY J2 - J GEN PHYSIOL VL - 155 PY - 2023 IS - 7 PG - 14 SN - 0022-1295 DO - 10.1085/jgp.202313352 UR - https://m2.mtmt.hu/api/publication/34007263 ID - 34007263 AB - Voltage-gated K+ channels have distinct gates that regulate ion flux: the activation gate (A-gate) formed by the bundle crossing of the S6 transmembrane helices and the slow inactivation gate in the selectivity filter. These two gates are bidirectionally coupled. If coupling involves the rearrangement of the S6 transmembrane segment, then we predict state-dependent changes in the accessibility of S6 residues from the water-filled cavity of the channel with gating. To test this, we engineered cysteines, one at a time, at S6 positions A471, L472, and P473 in a T449A Shaker-IR background and determined the accessibility of these cysteines to cysteine-modifying reagents MTSET and MTSEA applied to the cytosolic surface of inside-out patches. We found that neither reagent modified either of the cysteines in the closed or the open state of the channels. On the contrary, A471C and P473C, but not L472C, were modified by MTSEA, but not by MTSET, if applied to inactivated channels with open A-gate (OI state). Our results, combined with earlier studies reporting reduced accessibility of residues I470C and V474C in the inactivated state, strongly suggest that the coupling between the A-gate and the slow inactivation gate is mediated by rearrangements in the S6 segment. The S6 rearrangements are consistent with a rigid rod-like rotation of S6 around its longitudinal axis upon inactivation. S6 rotation and changes in its environment are concomitant events in slow inactivation of Shaker KV channels. LA - English DB - MTMT ER - TY - JOUR AU - Coonen, Laura AU - Martinez-Morales, Evelyn AU - Van De Sande, Dieter V. AU - Snyders, Dirk J. AU - Cortes, D. Marien AU - Cuello, Luis G. AU - Labro, Alain J. TI - The nonconducting W434F mutant adopts upon membrane depolarization an inactivated-like state that differs from wild-type Shaker-IR potassium channels JF - SCIENCE ADVANCES J2 - SCI ADV VL - 8 PY - 2022 IS - 37 PG - 11 SN - 2375-2548 DO - 10.1126/sciadv.abn1731 UR - https://m2.mtmt.hu/api/publication/33172175 ID - 33172175 AB - Voltage-gated K+ (Kv) channels mediate the flow of K+ across the cell membrane by regulating the conductive state of their activation gate (AG). Several Kv channels display slow C-type inactivation, a process whereby their selectivity filter (SF) becomes less or nonconductive. It has been proposed that, in the fast inactivation-removed Shaker-IR channel, the W434F mutation epitomizes the C-type inactivated state because it functionally accelerates this process. By introducing another pore mutation that prevents AG closure, P475D, we found a way to record ionic currents of the Shaker-IR-W434F-P475D mutant at hyperpolarized membrane potentials as the W434F-mutant SF recovers from its inactivated state. This W434F conductive state lost its high K+ over Na+ selectivity, and even NMDG+ can permeate, features not observed in a wild-type SF. This indicates that, at least during recovery from inactivation, the W434F-mutant SF transitions to a widened and noncationic specific conformation. LA - English DB - MTMT ER - TY - JOUR AU - Ong, Seow Theng AU - Tyagi, Anu AU - Chandy, K. George AU - Bhushan, Shashi TI - Mechanisms Underlying C-type Inactivation in Kv Channels: Lessons From Structures of Human Kv1.3 and Fly Shaker-IR Channels JF - FRONTIERS IN PHARMACOLOGY J2 - FRONT PHARMACOL VL - 13 PY - 2022 PG - 10 SN - 1663-9812 DO - 10.3389/fphar.2022.924289 UR - https://m2.mtmt.hu/api/publication/33158910 ID - 33158910 AB - Voltage-gated potassium (Kv) channels modulate the function of electrically-excitable and non-excitable cells by using several types of "gates" to regulate ion flow through the channels. An important gating mechanism, C-type inactivation, limits ion flow by transitioning Kv channels into a non-conducting inactivated state. Here, we highlight two recent papers, one on the human Kv1.3 channel and the second on the Drosophila Shaker Kv channel, that combined cryogenic electron microscopy and molecular dynamics simulation to define mechanisms underlying C-type inactivation. In both channels, the transition to the non-conducting inactivated conformation begins with the rupture of an intra-subunit hydrogen bond that fastens the selectivity filter to the pore helix. The freed filter swings outwards and gets tethered to an external residue. As a result, the extracellular end of the selectivity filter dilates and K+ permeation through the pore is impaired. Recovery from inactivation may entail a reversal of this process. Such a reversal, at least partially, is induced by the peptide dalazatide. Binding of dalazatide to external residues in Kv1.3 frees the filter to swing inwards. The extracellular end of the selectivity filter narrows allowing K+ to move in single file through the pore typical of conventional knock-on conduction. Inter-subunit hydrogen bonds that stabilize the outer pore in the dalazatide-bound structure are equivalent to those in open-conducting conformations of Kv channels. However, the intra-subunit bond that fastens the filter to the pore-helix is absent, suggesting an incomplete reversal of the process. These mechanisms define how Kv channels self-regulate the flow of K+ by changing the conformation of the selectivity filter. LA - English DB - MTMT ER - TY - JOUR AU - Bassetto, Carlos A. Z. AU - Carvalho-de-Souza, Joao Luis AU - Bezanilla, Francisco TI - Molecular basis for functional connectivity between the voltage sensor and the selectivity filter gate in Shaker K+ channels JF - ELIFE J2 - ELIFE VL - 10 PY - 2021 SN - 2050-084X DO - 10.7554/eLife.63077 UR - https://m2.mtmt.hu/api/publication/32029051 ID - 32029051 AB - In Shaker K+ channels, the S4-S5 linker couples the voltage sensor (VSD) and pore domain (PD). Another coupling mechanism is revealed using two W434F-containing channels: L361R:W434F and L366H:W434F. In L361R:W434F, W434F affects the L361R VSD seen as a shallower charge-voltage (Q-V) curve that crosses the conductance-voltage (G-V) curve. In L366H: W434F, L366H relieves the W434F effect converting a non-conductive channel in a conductive one. We report a chain of residues connecting the VSD (S4) to the selectivity filter (SF) in the PD of an adjacent subunit as the molecular basis for voltage sensor selectivity filter gate (VS-SF) coupling. Single alanine substitutions in this region (L409A, S411A, S412A, or F433A) are enough to disrupt the VS-SF coupling, shown by the absence of Q-V and G-V crossing in L361R:W434F mutant and by the lack of ionic conduction in the L366H:W434F mutant. This residue chain defines a new coupling between the VSD and the PD in voltage-gated channels. LA - English DB - MTMT ER - TY - JOUR AU - Li, Jing AU - Shen, Rong AU - Rohaim, Ahmed AU - Uriarte, Ramon Mendoza AU - Fajer, Mikolai AU - Perozo, Eduardo AU - Roux, Benoit TI - Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel JF - JOURNAL OF GENERAL PHYSIOLOGY J2 - J GEN PHYSIOL VL - 153 PY - 2021 IS - 9 PG - 19 SN - 0022-1295 DO - 10.1085/jgp.202112875 UR - https://m2.mtmt.hu/api/publication/32727945 ID - 32727945 AB - C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K center dot channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K center dot channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-mu s MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D. LA - English DB - MTMT ER - TY - JOUR AU - Szántó, Gábor Tibor AU - Gaál, Szabolcs Máté AU - Karbat, Izhar AU - Varga, Zoltán AU - Reuveny, Eitan AU - Panyi, György TI - Shaker-IR K+ channel gating in heavy water: Role of structural water molecules in inactivation JF - JOURNAL OF GENERAL PHYSIOLOGY J2 - J GEN PHYSIOL VL - 153 PY - 2021 IS - 6 PG - 20 SN - 0022-1295 DO - 10.1085/jgp.202012742 UR - https://m2.mtmt.hu/api/publication/32096927 ID - 32096927 AB - It has been reported earlier that the slow (C-type) inactivated conformation in Kv channels is stabilized by a multipoint hydrogen-bond network behind the selectivity filter. Furthermore, MD simulations revealed that structural water molecules are also involved in the formation of this network locking the selectivity filter in its inactive conformation. We found that the application of an extracellular, but not intracellular, solution based on heavy water (D2O) dramatically slowed entry into the slow inactivated state in Shaker-IR mutants (T449A, T449A/I470A, and T449K/I470C, displaying a wide range of inactivation kinetics), consistent with the proposed effect of the dynamics of structural water molecules on the conformational stability of the selectivity filter. Alternative hypotheses capable of explaining the observed effects of D2O were examined. Increased viscosity of the external solution mimicked by the addition of glycerol had a negligible effect on the rate of inactivation. In addition, the inactivation time constants of K+ currents in the outward and the inward directions in asymmetric solutions were not affected by a H2O/D2O exchange, negating an indirect effect of D2O on the rate of K+ rehydration. The elimination of the nonspecific effects of D2O on our macroscopic current measurements supports the hypothesis that the rate of structural water exchange at the region behind the selectivity filter determines the rate of slow inactivation, as proposed by molecular modeling. LA - English DB - MTMT ER - TY - JOUR AU - Nirenberg, Valerie Abigail AU - Yifrach, Ofer TI - Flow and shortcuts along the Shaker Kv channel slow inactivation gating cycle JF - JOURNAL OF GENERAL PHYSIOLOGY J2 - J GEN PHYSIOL VL - 152 PY - 2020 IS - 8 PG - 5 SN - 0022-1295 DO - 10.1085/jgp.202012611 UR - https://m2.mtmt.hu/api/publication/31739276 ID - 31739276 LA - English DB - MTMT ER -