A mutation affecting symbiosis in the pea line Risnod27 changes the ion selectivity filter of the DMI1 homolog

Activity of cyclic nucleotide–gated (CNG) cation channels underlies signal transduction in vertebrate visual receptors. These highly specialized receptor channels open when they bind cyclic GMP (cGMP). Here, we find that certain mutations restricted to the region around the ion selectivity filter render the channels essentially fully voltage gated, in such a manner that the channels remain mostly closed at physiological voltages, even in the presence of saturating concentrations of cGMP. This voltage-dependent gating resembles the selectivity filter-based mechanism seen in KcsA K+ channels, not the S4-based mechanism of voltage-gated K+ channels. Mutations that render CNG channels gated by voltage loosen the attachment of the selectivity filter to its surrounding structure, thereby shifting the channel's gating equilibrium toward closed conformations. Significant pore opening in mutant channels occurs only when positive voltages drive the pore from a low-probability open conformation toward a second open conformation to increase the channels' open probability. Thus, the structure surrounding the selectivity filter has evolved to (nearly completely) suppress the expression of inherent voltage-dependent gating of CNGA1, ensuring that the binding of cGMP by itself suffices to open the channels at physiological voltages.

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Ion-Selectivity Filter Interactions in KCSA from 1D 87Rb+ NMR

Biophysical Journal ◽

10.1016/j.bpj.2013.11.3010 ◽

2014 ◽

Vol 106 (2) ◽

pp. 540a

Author(s):

Raymond E. Hulse ◽

Joseph R. Sachleben ◽

Eduardo Perozo

Keyword(s):

Ion Selectivity ◽

Selectivity Filter

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Regulation of Kir Channels by Intracellular pH and Extracellular K+

The Journal of General Physiology ◽

10.1085/jgp.200308989 ◽

2004 ◽

Vol 123 (4) ◽

pp. 441-454 ◽

Cited By ~ 27

Author(s):

Anke Dahlmann ◽

Min Li ◽

ZhongHua Gao ◽

Deirdre McGarrigle ◽

Henry Sackin ◽

...

Keyword(s):

Ion Selectivity ◽

Transmembrane Helix ◽

Ph Sensor ◽

Outer Part ◽

Selectivity Filter ◽

Inward Rectification ◽

Ion Binding ◽

Enhanced Sensitivity ◽

Internal Ph ◽

Additional Channel

ROMK channels are regulated by internal pH (pHi) and extracellular K+ (K+o). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H+, increasing the apparent pKa for inhibition by ∼0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K+o, consistent with coupling between the responses to pHi and K+o. The ion selectivity sequence of the activation of the channel by cations was K+ ≅ Rb+ > NH4+ >> Na+, similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K+ from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K+o. We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K+o between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K+ to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba2+ block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H+. We conclude that decreasing pHI increases the sensitivity of ROMK2 channels to K+o by altering the properties of the selectivity filter.

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Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1609939113 ◽

2016 ◽

Vol 113 (48) ◽

pp. 13762-13767 ◽

Cited By ~ 25

Author(s):

Monica N. Kinde ◽

Vasyl Bondarenko ◽

Daniele Granata ◽

Weiming Bu ◽

Kimberly C. Grasty ◽

...

Keyword(s):

Sodium Channel ◽

Binding Sites ◽

Ion Selectivity ◽

Selectivity Filter ◽

Inactivation Kinetics ◽

Voltage Gated Sodium Channel ◽

Voltage Gated ◽

Voltage Gated Sodium Channels ◽

Binding Data ◽

Quantitative Analyses

Voltage-gated sodium channels (NaV) play an important role in general anesthesia. Electrophysiology measurements suggest that volatile anesthetics such as isoflurane inhibit NaVby stabilizing the inactivated state or altering the inactivation kinetics. Recent computational studies suggested the existence of multiple isoflurane binding sites in NaV, but experimental binding data are lacking. Here we use site-directed placement of19F probes in NMR experiments to quantify isoflurane binding to the bacterial voltage-gated sodium channel NaChBac.19F probes were introduced individually to S129 and L150 near the S4–S5 linker, L179 and S208 at the extracellular surface, T189 in the ion selectivity filter, and all phenylalanine residues. Quantitative analyses of19F NMR saturation transfer difference (STD) spectroscopy showed a strong interaction of isoflurane with S129, T189, and S208; relatively weakly with L150; and almost undetectable with L179 and phenylalanine residues. An orientation preference was observed for isoflurane bound to T189 and S208, but not to S129 and L150. We conclude that isoflurane inhibits NaChBac by two distinct mechanisms: (i) as a channel blocker at the base of the selectivity filter, and (ii) as a modulator to restrict the pivot motion at the S4–S5 linker and at a critical hinge that controls the gating and inactivation motion of S6.

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Permeability Properties of Enac Selectivity Filter Mutants

The Journal of General Physiology ◽

10.1085/jgp.118.6.679 ◽

2001 ◽

Vol 118 (6) ◽

pp. 679-692 ◽

Cited By ~ 58

Author(s):

Stephan Kellenberger ◽

Muriel Auberson ◽

Ivan Gautschi ◽

Estelle Schneeberger ◽

Laurent Schild

Keyword(s):

Amino Acid ◽

Transmembrane Domain ◽

Ion Selectivity ◽

Critical Role ◽

Selectivity Filter ◽

Side Chain ◽

Na Channel ◽

Ionic Selectivity ◽

Channel Pore ◽

Subunit Interface

The epithelial Na+ channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na+ absorption. The amiloride-sensitive ENaC is highly selective for Na+ and Li+ ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the αS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K+ ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted αS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position α589, indicating that αS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na+ and Li+. These findings demonstrate the critical role of the pore size at the αS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the αS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the αS589 side chain is oriented toward the subunit–subunit interface and that substitution of αS589 by larger residues increases the pore diameter by adding extra volume at the subunit–subunit interface.

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An Ion Selectivity Filter in the Extracellular Domain of Cys-loop Receptors Reveals Determinants for Ion Conductance

Journal of Biological Chemistry ◽

10.1074/jbc.c800194200 ◽

2008 ◽

Vol 283 (52) ◽

pp. 36066-36070 ◽

Cited By ~ 46

Author(s):

Scott B. Hansen ◽

Hai-Long Wang ◽

Palmer Taylor ◽

Steven M. Sine

Keyword(s):

Ion Selectivity ◽

Extracellular Domain ◽

Selectivity Filter ◽

Ion Conductance

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Conformational dynamics in TRPV1 channels reported by an encoded coumarin amino acid

10.1101/137778 ◽

2017 ◽

Author(s):

Ximena Steinberg ◽

Marina A. Kasimova ◽

Deny Cabezas-Bratesco ◽

Jason Galpin ◽

Ernesto Ladrón-de-Guevara ◽

...

Keyword(s):

Amino Acid ◽

Ion Selectivity ◽

Noise Analysis ◽

Conformational Dynamics ◽

Selectivity Filter ◽

Optical Data ◽

Transient Receptor Potential Vanilloid ◽

Solvent Exposure ◽

Channel Activation ◽

Trpv1 Channels

ABSTRACTTransient Receptor Potential Vanilloid (TRPV1) channels support the detection and integration of nociceptive input. Currently available functional and structural data suggest that that TRPV1 channels have two potential gates within their cation selective permeation pathway: a barrier formed by a ‘bundle crossing’ at the intracellular entrance and a second constriction created by the ion selectivity filter. To describe conformational changes associated with channel gating within the pore, the fluorescent non-canonical amino acid (f- ncAA) coumarin-tyrosine was genetically encoded at Y671, a residue proximal to the selectivity filter. TRPV1 channels expressing coumarin at either site displayed normal voltage- and agonist-dependent gating. Next, total internal reflection microscopy (TIRF) was performed to enable ultra-rapid, millisecond imaging of the conformational dynamics in single TRPV1 channels in live cells. Here, the data obtained from channels expressed in human derived cells show that optical fluctuations, photon counts, and variance of noise analysis from Y671 coumarin encoded in TRPV1 tetramers correlates closely with channel activation by capsaicin, thus providing an direct optical marker of channel activation at the selectivity filter. In companion molecular dynamics simulations, Y671 displays alternating solvent exposure between the closed and open states, giving support to the optical data. These calculations further suggest a direct involvement of Y671 in controlling the relative position of the pore helix and its role in supporting ionic conductance at the TRPV1 selectivity filter.

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Mechanism of the Voltage Sensitivity of IRK1 Inward-rectifier K+ Channel Block by the Polyamine Spermine

The Journal of General Physiology ◽

10.1085/jgp.200409242 ◽

2005 ◽

Vol 125 (4) ◽

pp. 413-426 ◽

Cited By ~ 45

Author(s):

Hyeon-Gyu Shin ◽

Zhe Lu

Keyword(s):

Steady State ◽

Voltage Dependence ◽

Ion Selectivity ◽

Inward Rectifier ◽

Selectivity Filter ◽

K Channel ◽

Voltage Sensitivity ◽

Channel Block ◽

Head Groups ◽

Voltage Dependent

IRK1 (Kir2.1) inward-rectifier K+ channels exhibit exceedingly steep rectification, which reflects strong voltage dependence of channel block by intracellular cations such as the polyamine spermine. On the basis of studies of IRK1 block by various amine blockers, it was proposed that the observed voltage dependence (valence ∼5) of IRK1 block by spermine results primarily from K+ ions, not spermine itself, traversing the transmembrane electrical field that drops mostly across the narrow ion selectivity filter, as spermine and K+ ions displace one another during channel block and unblock. If indeed spermine itself only rarely penetrates deep into the ion selectivity filter, then a long blocker with head groups much wider than the selectivity filter should exhibit comparably strong voltage dependence. We confirm here that channel block by two molecules of comparable length, decane-bis-trimethylammonium (bis-QAC10) and spermine, exhibit practically identical overall voltage dependence even though the head groups of the former are much wider (∼6 Å) than the ion selectivity filter (∼3 Å). For both blockers, the overall equilibrium dissociation constant differs from the ratio of apparent rate constants of channel unblock and block. Also, although steady-state IRK1 block by both cations is strongly voltage dependent, their apparent channel-blocking rate constant exhibits minimal voltage dependence, which suggests that the pore becomes blocked as soon as the blocker encounters the innermost K+ ion. These findings strongly suggest the existence of at least two (potentially identifiable) sequentially related blocked states with increasing numbers of K+ ions displaced. Consequently, the steady-state voltage dependence of IRK1 block by spermine or bis-QAC10 should increase with membrane depolarization, a prediction indeed observed. Further kinetic analysis identifies two blocked states, and shows that most of the observed steady-state voltage dependence is associated with the transition between blocked states, consistent with the view that the mutual displacement of blocker and K+ ions must occur mainly as the blocker travels along the long inner pore.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1515965112 ◽

2015 ◽

Vol 112 (50) ◽

pp. 15366-15371 ◽

Cited By ~ 8

Author(s):

Joshua B. Brettmann ◽

Darya Urusova ◽

Marco Tonelli ◽

Jonathan R. Silva ◽

Katherine A. Henzler-Wildman

Keyword(s):

Ion Selectivity ◽

Selectivity Filter ◽

Structural Basis ◽

Functional Coupling ◽

Dynamic Coupling ◽

Allosteric Coupling ◽

Allosteric Communication ◽

Dependent Inactivation ◽

Ion Binding Sites ◽

Essential Connection

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 potassium-selective 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.

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