scholarly journals Conformational changes upon gating of KirBac1.1 into an open-activated state revealed by solid-state NMR and functional assays

2020 ◽  
Vol 117 (6) ◽  
pp. 2938-2947 ◽  
Author(s):  
Reza Amani ◽  
Collin G. Borcik ◽  
Nazmul H. Khan ◽  
Derek B. Versteeg ◽  
Maryam Yekefallah ◽  
...  
Keyword(s):  
Solid State ◽  
Conformational Changes ◽  
Solid State Nmr ◽  
Lipid Binding ◽  
Selectivity Filter ◽  
Conformational Exchange ◽  
Lipid Mixture ◽  
Kir Channels ◽  
Activated State ◽  
Activation Gate

The conformational changes required for activation and K+ conduction in inward-rectifier K+ (Kir) channels are still debated. These structural changes are brought about by lipid binding. It is unclear how this process relates to fast gating or if the intracellular and extracellular regions of the protein are coupled. Here, we examine the structural details of KirBac1.1 reconstituted into both POPC and an activating lipid mixture of 3:2 POPC:POPG (wt/wt). KirBac1.1 is a prokaryotic Kir channel that shares homology with human Kir channels. We establish that KirBac1.1 is in a constitutively active state in POPC:POPG bilayers through the use of real-time fluorescence quenching assays and Förster resonance energy transfer (FRET) distance measurements. Multidimensional solid-state NMR (SSNMR) spectroscopy experiments reveal two different conformers within the transmembrane regions of the protein in this activating lipid environment, which are distinct from the conformation of the channel in POPC bilayers. The differences between these three distinct channel states highlight conformational changes associated with an open activation gate and suggest a unique allosteric pathway that ties the selectivity filter to the activation gate through interactions between both transmembrane helices, the turret, selectivity filter loop, and the pore helix. We also identify specific residues involved in this conformational exchange that are highly conserved among human Kir channels.

scholarly journals Probing Allosteric Coupling of a Constitutively Open Mutant of the Ion Channel KcsA using Solid State NMR

2019 ◽  
Author(s):  
Zhiyu Sun ◽  
Yunyao Xu ◽  
Dongyu Zhang ◽  
Ann E McDermott
Keyword(s):  
Solid State ◽  
Binding Affinity ◽  
Solid State Nmr ◽  
Selectivity Filter ◽  
Wild Type ◽  
Allosteric Coupling ◽  
Channel Inactivation ◽  
Activation Gate ◽  
Inactivation Process ◽  
Potassium Binding

AbstractTransmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the mean open time of the prototypical potassium channel KcsA in the context of C-type inactivation. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. We tested this mechanism for inactivation using a KcsA mutant (H25R/E118A) that has the pH gate open across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. The potassium binding affinity in the selectivity filter of this mutant, 81 mM, is about 4 orders of magnitude weaker than that of wild type KcsA at neutral pH and is comparable to the value for wild type KcsA at low pH (pH ∼ 3.5). This result strongly supports our assertion that the open pH gate allosterically effects the potassium binding affinity of the selectivity filter. In this mutant the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery.Significance statementInactivation of potassium channels controls mean open times and provides exquisite control over biological processes. In the highly conserved C-type inactivation process, opening of the activation gate causes subsequent inactivation. We test whether the open state of the channel simply has a poor ability to bind the K+ ion. Previously, activated and inactivated states were stabilized using truncations or a significant pH drop. Here, we use the H25R/E118A constitutively open mutant of KcsA and also observe a large drop in potassium binding affinity. This provides strong evidence that channel opening causes an allosteric loss of ion affinity, and that the central feature of this universal channel inactivation process is loss of ion affinity at the selectivity filter.

scholarly journals Probing allosteric coupling in a constitutively open mutant of the ion channel KcsA using solid-state NMR

2020 ◽  
Vol 117 (13) ◽  
pp. 7171-7175
Author(s):  
Zhiyu Sun ◽  
Yunyao Xu ◽  
Dongyu Zhang ◽  
Ann E. McDermott
Keyword(s):  
Solid State ◽  
Binding Affinity ◽  
Solid State Nmr ◽  
Selectivity Filter ◽  
Wild Type ◽  
Proton Binding ◽  
Allosteric Coupling ◽  
Neutral Ph ◽  
Potassium Channel Kcsa ◽  
Potassium Binding

Transmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the potassium binding affinity of the prototypical potassium channel KcsA in the context of C-type inactivation. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch, leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. We tested this mechanism for inactivation using a KcsA mutant (H25R/E118A) that exhibits an open pH gate across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. The potassium binding affinity in the selectivity filter of this mutant, 81 mM, is about four orders of magnitude weaker than that of wild-type KcsA at neutral pH and is comparable to the value for wild-type KcsA at low pH (pH ≈ 3.5). This result strongly supports our assertion that the open pH gate allosterically affects the potassium binding affinity of the selectivity filter. In this mutant, the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery.

Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR

Nature ◽  
2006 ◽  
Vol 440 (7086) ◽  
pp. 959-962 ◽  
Author(s):  
Adam Lange ◽  
Karin Giller ◽  
Sönke Hornig ◽  
Marie-France Martin-Eauclaire ◽  
Olaf Pongs ◽  
...  
Keyword(s):  
Solid State ◽  
Potassium Channel ◽  
Conformational Changes ◽  
Solid State Nmr

scholarly journals 2P247 Photoactivated conformational changes of photoreceptor membrane protein ppR/pHtrII observed by in situ photo irradiation solid-state NMR(18A. Photobiology: Vision & Photoreception,Poster)

2013 ◽  
Vol 53 (supplement1-2) ◽  
pp. S199
Author(s):  
Yoshiteru Makino ◽  
Yuya Tomonaga ◽  
Yusuke Shibafuji ◽  
Tetsurou Hidaka ◽  
Izuru Kawamura ◽  
...  
Keyword(s):  
Solid State ◽  
Membrane Protein ◽  
Conformational Changes ◽  
Solid State Nmr ◽  
Photoreceptor Membrane

scholarly journals Nanotube Array Method for Studying Lipid-Induced Conformational Changes of a Membrane Protein by Solid-State NMR

2015 ◽  
Vol 108 (1) ◽  
pp. 5-9 ◽  
Author(s):  
Antonin Marek ◽  
Wenxing Tang ◽  
Sergey Milikisiyants ◽  
Alexander A. Nevzorov ◽  
Alex I. Smirnov
Keyword(s):  
Solid State ◽  
Membrane Protein ◽  
Conformational Changes ◽  
Solid State Nmr ◽  
Nanotube Array

scholarly journals Closed state-coupled C-type inactivation in BK channels

2016 ◽  
Vol 113 (25) ◽  
pp. 6991-6996 ◽  
Author(s):  
Jiusheng Yan ◽  
Qin Li ◽  
Richard W. Aldrich
Keyword(s):  
Conformational Changes ◽  
Bk Channels ◽  
Bk Channel ◽  
Membrane Potentials ◽  
Selectivity Filter ◽  
Open Probability ◽  
Closed State ◽  
State Dependent ◽  
Channel Opening ◽  
Activation Gate

Ion channels regulate ion flow by opening and closing their pore gates. K+ channels commonly possess two pore gates, one at the intracellular end for fast channel activation/deactivation and the other at the selectivity filter for slow C-type inactivation/recovery. The large-conductance calcium-activated potassium (BK) channel lacks a classic intracellular bundle-crossing activation gate and normally show no C-type inactivation. We hypothesized that the BK channel’s activation gate may spatially overlap or coexist with the C-type inactivation gate at or near the selectivity filter. We induced C-type inactivation in BK channels and studied the relationship between activation/deactivation and C-type inactivation/recovery. We observed prominent slow C-type inactivation/recovery in BK channels by an extreme low concentration of extracellular K+ together with a Y294E/K/Q/S or Y279F mutation whose equivalent in Shaker channels (T449E/K/D/Q/S or W434F) caused a greatly accelerated rate of C-type inactivation or constitutive C-inactivation. C-type inactivation in most K+ channels occurs upon sustained membrane depolarization or channel opening and then recovers during hyperpolarized membrane potentials or channel closure. However, we found that the BK channel C-type inactivation occurred during hyperpolarized membrane potentials or with decreased intracellular calcium ([Ca2+]i) and recovered with depolarized membrane potentials or elevated [Ca2+]i. Constitutively open mutation prevented BK channels from C-type inactivation. We concluded that BK channel C-type inactivation is closed state-dependent and that its extents and rates inversely correlate with channel-open probability. Because C-type inactivation can involve multiple conformational changes at the selectivity filter, we propose that the BK channel’s normal closing may represent an early conformational stage of C-type inactivation.

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