scholarly journals Regulation of Kir Channels by Intracellular pH and Extracellular K+

2004 ◽  
Vol 123 (4) ◽  
pp. 441-454 ◽  
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.

scholarly journals K2P channel C-type gating involves asymmetric selectivity filter order-disorder transitions

2020 ◽  
Author(s):  
Marco Lolicato ◽  
Andrew M. Natale ◽  
Fayal Abderemane-Ali ◽  
David Crottès ◽  
Sara Capponi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Ion Selectivity ◽  
Transmembrane Helix ◽  
Selectivity Filter ◽  
Ion Binding ◽  
Anomalous Scattering ◽  
X Ray Crystallography ◽  
Functional Studies ◽  
Ion Binding Sites ◽  
Physical And Chemical

K2P channels regulate nervous, cardiovascular, and immune system functions1,2 through the action of their selectivity filter (C-type) gate3-6. Although structural studies show K2P conformations that impact activity7-13, no selectivity filter conformational changes have been observed. Here, combining K2P2.1 (TREK-1) X-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics, and functional studies, we uncover the unprecedented, asymmetric, potassium-dependent conformational changes underlying K2P C-type gating. Low potassium concentrations evoke conformational changes in selectivity filter strand 1 (SF1), selectivity filter strand 2 (SF2), and the SF2-transmembrane helix 4 loop (SF2-M4 loop) that destroy the S1 and S2 ion binding sites and are suppressed by C-type gate activator ML335. Shortening the uniquely long SF2-M4 loop to match the canonical length found in other potassium channels or disrupting the conserved Glu234 hydrogen bond network supporting this loop blunts C-type gate response to various physical and chemical stimuli. Glu234 network destabilization also compromises ion selectivity, but can be reversed by channel activation, indicating that the ion binding site loss reduces selectivity similar to other channels14. Together, our data establish that C-type gating occurs through potassium-dependent order-disorder transitions in the selectivity filter and adjacent loops that respond to gating cues relayed through the SF2-M4 loop. These findings underscore the potential for targeting the SF2-M4 loop for the development of new, selective K2P channel modulators.

scholarly journals Pore-forming transmembrane domains control ion selectivity and selectivity filter conformation in the KirBac1.1 potassium channel

2021 ◽  
Vol 153 (5) ◽  
Author(s):  
Marcos Matamoros ◽  
Colin G. Nichols
Keyword(s):  
Single Molecule ◽  
Ion Selectivity ◽  
Transmembrane Helix ◽  
Selectivity Filter ◽  
K Channel ◽  
Physical Interaction ◽  
Conformationally Constrained ◽  
Single Molecule Fret ◽  
High K ◽  
Low K

Potassium (K+) channels are membrane proteins with the remarkable ability to very selectively conduct K+ ions across the membrane. High-resolution structures have revealed that dehydrated K+ ions permeate through the narrowest region of the pore, formed by the backbone carbonyls of the signature selectivity filter (SF) sequence TxGYG. However, the existence of nonselective channels with similar SF sequences, as well as effects of mutations in other regions on selectivity, suggest that the SF is not the sole determinant of selectivity. We changed the selectivity of the KirBac1.1 channel by introducing mutations at residue I131 in transmembrane helix 2 (TM2). These mutations increase Na+ flux in the absence of K+ and introduce significant proton conductance. Consistent with K+ channel crystal structures, single-molecule FRET experiments show that the SF is conformationally constrained and stable in high-K+ conditions but undergoes transitions to dilated low-FRET states in high-Na+/low-K+ conditions. Relative to wild-type channels, I131M mutants exhibit marked shifts in the K+ and Na+ dependence of SF dynamics to higher K+ and lower Na+ concentrations. These results illuminate the role of I131, and potentially other structural elements outside the SF, in controlling ion selectivity, by suggesting that the physical interaction of these elements with the SF contributes to the relative stability of the constrained K+-induced SF configuration versus nonselective dilated conformations.

scholarly journals Transmembrane allosteric energetics characterization for strong coupling between proton and potassium ion binding in the KcsA channel

2017 ◽  
Vol 114 (33) ◽  
pp. 8788-8793 ◽  
Author(s):  
Yunyao Xu ◽  
Manasi P. Bhate ◽  
Ann E. McDermott
Keyword(s):  
Ion Channel ◽  
Binding Site ◽  
Ph Sensor ◽  
Selectivity Filter ◽  
Ion Binding ◽  
Wild Type ◽  
Channel Activation ◽  
Site Specific ◽  
Allosteric Coupling ◽  
Allosteric Mechanism

The slow spontaneous inactivation of potassium channels exhibits classic signatures of transmembrane allostery. A variety of data support a model in which the loss of K+ ions from the selectivity filter is a major factor in promoting inactivation, which defeats transmission, and is allosterically coupled to protonation of key channel activation residues, more than 30 Å from the K+ ion binding site. We show that proton binding at the intracellular pH sensor perturbs the potassium affinity at the extracellular selectivity filter by more than three orders of magnitude for the full-length wild-type KcsA, a pH-gated bacterial channel, in membrane bilayers. Studies of F103 in the hinge of the inner helix suggest an important role for its bulky sidechain in the allosteric mechanism; we show that the energetic strength of coupling of the gates is strongly altered when this residue is mutated to alanine. These results provide quantitative site-specific measurements of allostery in a bilayer environment, and highlight the power of describing ion channel gating through the lens of allosteric coupling.

scholarly journals Structure, function, and ion-binding properties of a K+ channel stabilized in the 2,4-ion–bound configuration

2019 ◽  
Vol 116 (34) ◽  
pp. 16829-16834 ◽  
Author(s):  
Cholpon Tilegenova ◽  
D. Marien Cortes ◽  
Nermina Jahovic ◽  
Emily Hardy ◽  
Parameswaran Hariharan ◽  
...  
Keyword(s):  
Single Channel ◽  
Ion Selectivity ◽  
Streaming Potential ◽  
Point Of View ◽  
Selectivity Filter ◽  
K Channel ◽  
Crystallographic Structure ◽  
Ion Binding ◽  
Coupled Transport ◽  
Binding Properties

Here, we present the atomic resolution crystallographic structure, the function, and the ion-binding properties of the KcsA mutants, G77A and G77C, that stabilize the 2,4-ion–bound configuration (i.e., water, K+, water, K+-ion–bound configuration) of the K+ channel’s selectivity filter. A full functional and thermodynamic characterization of the G77A mutant revealed wild-type–like ion selectivity and apparent K+-binding affinity, in addition to showing a lack of C-type inactivation gating and a marked reduction in its single-channel conductance. These structures validate, from a structural point of view, the notion that 2 isoenergetic ion-bound configurations coexist within a K+ channel’s selectivity filter, which fully agrees with the water–K+-ion–coupled transport detected by streaming potential measurements.

scholarly journals Ion Interactions in the High-Affinity Binding Locus of a Voltage-Gated Ca2+ Channel

2000 ◽  
Vol 116 (4) ◽  
pp. 569-586 ◽  
Author(s):  
Robin K. Cloues ◽  
Susan M. Cibulsky ◽  
William A. Sather
Keyword(s):  
Binding Affinity ◽  
Single Channel ◽  
Ion Selectivity ◽  
Ion Pairs ◽  
Selectivity Filter ◽  
Ion Binding ◽  
Wild Type ◽  
Filter Function ◽  
Voltage Gated ◽  
Ca2 Channels

The selectivity filter of voltage-gated Ca2+ channels is in part composed of four Glu residues, termed the EEEE locus. Ion selectivity in Ca2+ channels is based on interactions between permeant ions and the EEEE locus: in a mixture of ions, all of which can pass through the pore when present alone, those ions that bind weakly are impermeant, those that bind more strongly are permeant, and those that bind more strongly yet act as pore blockers as a consequence of their low rate of unbinding from the EEEE locus. Thus, competition among ion species is a determining feature of selectivity filter function in Ca2+ channels. Previous work has shown that Asp and Ala substitutions in the EEEE locus reduce ion selectivity by weakening ion binding affinity. Here we describe for wild-type and EEEE locus mutants an analysis at the single channel level of competition between Cd2+, which binds very tightly within the EEEE locus, and Ba2+ or Li+, which bind less tightly and hence exhibit high flux rates: Cd2+ binds to the EEEE locus ∼104× more tightly than does Ba2+, and ∼108× more tightly than does Li+. For wild-type channels, Cd2+ entry into the EEEE locus was 400× faster when Li+ rather than Ba2+ was the current carrier, reflecting the large difference between Ba2+ and Li+ in affinity for the EEEE locus. For the substitution mutants, analysis of Cd2+ block kinetics shows that their weakened ion binding affinity can result from either a reduction in blocker on rate or an enhancement of blocker off rate. Which of these rate effects underlay weakened binding was not specified by the nature of the mutation (Asp vs. Ala), but was instead determined by the valence and affinity of the current-carrying ion (Ba2+ vs. Li+). The dependence of Cd2+ block kinetics upon properties of the current-carrying ion can be understood by considering the number of EEEE locus oxygen atoms available to interact with the different ion pairs.

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

2009 ◽  
Vol 53 (3) ◽  
pp. 451-460 ◽  
Author(s):  
K. Novak ◽  
J. Felsberg ◽  
E. Biedermannova ◽  
J. Vondrys
Keyword(s):  
Ion Selectivity ◽  
Selectivity Filter

scholarly journals Tryptophan 207 is crucial to the unique properties of the human voltage-gated proton channel, hHV1

2015 ◽  
Vol 146 (5) ◽  
pp. 343-356 ◽  
Author(s):  
Vladimir V. Cherny ◽  
Deri Morgan ◽  
Boris Musset ◽  
Gustavo Chaves ◽  
Susan M.E. Smith ◽  
...  
Keyword(s):  
Transmembrane Helix ◽  
Ph Sensor ◽  
Proton Channel ◽  
Tryptophan Residue ◽  
Ph Sensing ◽  
Ph Sensors ◽  
Voltage Gated ◽  
Karlodinium Veneficum ◽  
Channel Opening ◽  
External Ph

Part of the “signature sequence” that defines the voltage-gated proton channel (HV1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence HV1 genes. Replacing Trp207 in human HV1 (hHV1) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, Ea, for channel opening decreased to 22 kcal/mol from 30–38 kcal/mol for WT, confirming that Trp207 establishes the major energy barrier between closed and open hHV1. Cation–π interaction between Trp207 and Arg211 evidently latches the channel closed. Trp207 mutants lost proton selectivity at pHo >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of HV1 that is essential to its biological functions, was compromised. In the WT hHV1, ΔpH-dependent gating is shown to saturate above pHi or pHo 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp207 mutants, ΔpH-dependent gating saturated at lower pHo but not at lower pHi. That Trp207 mutation selectively alters pHo sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in HV1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pHo and pHi in HV1 of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating.

Ion Binding to KcsA: Implications in Ion Selectivity and Channel Gating

Biochemistry ◽  
2010 ◽  
Vol 49 (44) ◽  
pp. 9480-9487 ◽  
Author(s):  
M. L. Renart ◽  
I. Triano ◽  
J. A. Poveda ◽  
J. A. Encinar ◽  
A. M. Fernández ◽  
...  
Keyword(s):  
Ion Selectivity ◽  
Channel Gating ◽  
Ion Binding

Mutations in the pore region of ROMK enhance Ba2+ block

1996 ◽  
Vol 271 (6) ◽  
pp. C1949-C1956 ◽  
Author(s):  
H. Zhou ◽  
S. Chepilko ◽  
W. Schutt ◽  
H. Choe ◽  
L. G. Palmer ◽  
...  
Keyword(s):  
Single Channel ◽  
Ion Selectivity ◽  
Inward Rectification ◽  
Xenopus Laevis Oocytes ◽  
Channel Measurements ◽  
Pore Region ◽  
Selective Channel ◽  
Increased Sensitivity ◽  
Membrane Spanning ◽  
Zero Voltage

The sequence of the hydrophobic “P” (pore) region of a K(+)-selective channel from the kidney (ROMK2) was altered to match that of the closely related inward rectifier (IRK1) channel by changing two amino acids, leucine (L) 117 and valine (V) 121, to isoleucine (I) and threonine (T), respectively. The mutant channel expressed in Xenopus laevis oocytes had an apparent inhibition constant at zero voltage [Ki(0)] in the presence of Ba2+ of 0.07 +/- 0.01 mM, which was more than 50 times lower than the Ki(0) of the wild-type channel (4.7 +/- 1.0 mM). The increased sensitivity to Ba2+ was accounted for by the point mutation V121T. Single-channel measurements indicated that the increased affinity involved an increase in the on-rate for Ba2+ block and a decrease in the off-rate. Block by Ca+ was also enhanced. The single-channel conductance of the L1171/ V121T mutant was increased by 50%, whereas the degree of inward rectification, ion selectivity, and apparent affinity for K+ were essentially unchanged. When the neutral asparagine residue within the second putative membrane-spanning domain of the ROMK channel was substituted with aspartic acid, the corresponding amino acid in IRK1, the degree of inward rectification was enhanced but Ba2+ block and single-channel inward conductance were unaffected. Thus the site of Ba2+ binding appears to be distinct from the locus of internal Mg2+ block and from at least one of the sites that determines K+ conjuctivity.

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