Functional Equilibrium of the KcsA Structure Revealed by NMR

Imai, Shunsuke; Osawa, Masatoshi; Mita, Kenichiro; Toyonaga, Shou; Machiyama, Asako; Ueda, Takumi; Takeuchi, Koh; Oiki, Shigetoshi; Shimada, Ichio

doi:10.1074/jbc.m112.401265

Cited by 49 publications

(60 citation statements)

References 28 publications

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“…To correlate the changes in P O with conformational changes at the channel pore, we performed two-dimensional (2D) ( 13 C, 13 C) and ( 15 N, 13 C) ssNMR correlation experiments on KcsA in phospholipid environments identical to those used in the electrophysiological experiments described above. In contrast to previous solution-state NMR experiments using bilayer mimetics (see, e.g., (42)(43)(44)), the 13 C-13 C correlation spectra recorded with short mixing times enabled us to identify 13 C chemical shifts (see Table S2 for a complete list) for the key residues T74, T75, and V76 within the KcsA selectivity filter (Fig. 2, A and B), as well as for G99, I100, and T101 located at the activation gate (Fig.…”

Section: Resultsmentioning

confidence: 84%

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Cruijsen

Prokofyev

Pongs

et al. 2017

Biophysical Journal

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Ion conduction across the cellular membrane requires the simultaneous opening of activation and inactivation gates of the K þ channel pore. The bacterial KcsA channel has served as a powerful system for dissecting the structural changes that are related to four major functional states associated with K þ gating. Yet, the direct observation of the full gating cycle of KcsA has remained structurally elusive, and crystal structures mimicking these gating events require mutations in or stabilization of functionally relevant channel segments. Here, we found that changes in lipid composition strongly increased the KcsA open probability. This enabled us to probe all four major gating states in native-like membranes by combining electrophysiological and solid-state NMR experiments. In contrast to previous crystallographic views, we found that the selectivity filter and turret region, coupled to the surrounding bilayer, were actively involved in channel gating. The increase in overall steady-state open probability was accompanied by a reduction in activation-gate opening, underscoring the important role of the surrounding lipid bilayer in the delicate conformational coupling of the inactivation and activation gates.

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Section: Resultsmentioning

confidence: 84%

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Cruijsen

Prokofyev

Pongs

et al. 2017

Biophysical Journal

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“…The rHDLs reportedly provide a lipid environment with more native-like properties, compared with liposomes, in terms of the lateral pressure and curvature profiles because detergent micelles have strong curvature and different lateral pressure profiles from lipid membranes (31). Our NMR analyses of a G protein-coupled receptor (GPCR) and an ion channel in rHDL lipid bilayers revealed that the population and the exchange rates of the conformational equilibrium determine their signal transduction and ion transport activities (32)(33)(34) and that the population of the active conformation of the GPCR in rHDLs correlated better with the signaling levels than that in detergent micelles (32). Therefore, NMR investigations of membrane proteins in the lipid bilayer environments of rHDLs are necessary for accurate measurements of the exchange rates and the populations in conformational equilibrium.…”

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confidence: 99%

Conductance of P2X ₄ purinergic receptor is determined by conformational equilibrium in the transmembrane region

Minato

Suzuki

Hara

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cationselective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X 4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X 4 purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s −1 ), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.NMR | membrane proteins | ligand-gated ion channels | insect cell expression system | purinergic receptors I n chemical neurotransmission, various neurotransmitters bind to ligand-gated ion channels expressed in the plasma membrane of postsynaptic cells, such as the NMDA, AMPA, and P2X receptors, leading to changes in membrane potential and the concentration of intracellular ions. Each ligand for a ligand-gated ion channel has a distinct ability to evoke currents (1), and the ligands are classified according to the evoked current level: such as, full agonists, partial agonists, and antagonists. Partial agonists of ligand-gated ion channels reportedly offer clinical advantages over antagonists and full agonists in antidepressant and smoking-cessation treatment (2, 3).Two mechanisms have been proposed for the partial activation of the ligand-gated ion channels: the equilibrium between the open and closed conformations and the distinct conformation of the partial agonist-bound states from the closed and open conformations (4, 5). In the crystal structures of the extracellular region of the AMPA receptor, in which the distances between the two extracellular domains are changed upon agonist binding, the interdomain distances in the partial agonist-bound states correlated with the conductance level, suggesting that the AMPA receptor adopts specific intermediately permeable conformations (4, 6).The P2X receptors are a family of cation channe...

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“…These two gates are functionally coupled as demonstrated by C-type inactivation, in which channel opening triggers loss of conduction at the selectivity filter (1)(2)(3)(4). A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4)(5)(6)(7)(8)(9)(10). In the reverse pathway, inactivation of the selectivity filter has been linked to changes at the inner gate (5)(6)(7)(8)(9)(10)(11)(12)(13)(14).…”

mentioning

confidence: 99%

“…A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4)(5)(6)(7)(8)(9)(10). In the reverse pathway, inactivation of the selectivity filter has been linked to changes at the inner gate (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). However, flux-dependent inactivation occurs in Na + and Ca 2+ channels as well and would likely require a structurally different mechanism to explain coupling between the selectivity filter and inner gate (7,(13)(14)(15)(16)(17)(18).…”

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confidence: 99%

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

Brettmann

Urusova

Tonelli

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassiumselective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.solution NMR | ion channels | membrane protein | protein dynamics

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Functional Equilibrium of the KcsA Structure Revealed by NMR

Cited by 49 publications

References 28 publications

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Conductance of P2X ₄ purinergic receptor is determined by conformational equilibrium in the transmembrane region

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

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Functional Equilibrium of the KcsA Structure Revealed by NMR

Cited by 49 publications

References 28 publications

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Conductance of P2X 4 purinergic receptor is determined by conformational equilibrium in the transmembrane region

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

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Conductance of P2X ₄ purinergic receptor is determined by conformational equilibrium in the transmembrane region