Ion-binding properties of a K+ channel selectivity filter in different conformations

K+ channels are membrane proteins that selectively conduct K+ ions across lipid bilayers. Many voltage-gated K+ (KV) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K+ channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to KV channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na+ or low [K+]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K+ ion binding to channels with an inactivated or conductive selectivity filter is different from K+ ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1901888116 ◽

2019 ◽

Vol 116 (34) ◽

pp. 16829-16834 ◽

Cited By ~ 6

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.

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A minimalist model for ion partitioning and competition in a K+ channel selectivity filter

The Journal of General Physiology ◽

10.1085/jgp.201110694 ◽

2011 ◽

Vol 138 (3) ◽

pp. 371-373 ◽

Cited By ~ 6

Author(s):

Stefan M. Kast ◽

Thomas Kloss ◽

Sascha Tayefeh ◽

Gerhard Thiel

Keyword(s):

Selectivity Filter ◽

K Channel ◽

Ion Partitioning ◽

Channel Selectivity

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Mechanism of charybdotoxin block of the high-conductance, Ca2+-activated K+ channel.

The Journal of General Physiology ◽

10.1085/jgp.91.3.335 ◽

1988 ◽

Vol 91 (3) ◽

pp. 335-349 ◽

Cited By ~ 250

Author(s):

R MacKinnon ◽

C Miller

Keyword(s):

Lipid Bilayers ◽

External Solution ◽

Dissociation Rate ◽

K Channel ◽

Internal Solution ◽

Conduction Pathway ◽

Voltage Dependent ◽

K Concentration ◽

A Site ◽

Low K

The mechanism of charybdotoxin (CTX) block of single Ca2+-activated K+ channels from rat muscle was studied in planar lipid bilayers. CTX blocks the channel from the external solution, and K+ in the internal solution specifically relieves toxin block. The effect of K+ is due solely to an enhancement of the CTX dissociation rate. As internal K+ is raised, the CTX dissociation rate increases in a rectangular hyperbolic fashion from a minimum value at low K+ of 0.01 s-1 to a maximum value of approximately 0.2 s-1. As the membrane is depolarized, internal K+ more effectively accelerates CTX dissociation. As the membrane is hyperpolarized, the toxin dissociation rate approaches 0.01 s-1, regardless of the K+ concentration. When internal K+ is replaced by Na+, CTX dissociation is no longer voltage dependent. The permeant ion Rb also accelerates toxin dissociation from the internal solution, while the impermeant ions Li, Na, Cs, and arginine do not. These results argue that K ions can enter the CTX-blocked channel from the internal solution to reach a site located nearly all the way through the conduction pathway; when K+ occupies this site, CTX is destabilized on its blocking site by approximately 1.8 kcal/mol. The most natural way to accommodate these conclusions is to assume that CTX physically plugs the channel's externally facing mouth.

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Initial steps of inactivation at the K+ channel selectivity filter

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1317573111 ◽

2014 ◽

Vol 111 (17) ◽

pp. E1713-E1722 ◽

Cited By ~ 13

Author(s):

A. S. Thomson ◽

F. T. Heer ◽

F. J. Smith ◽

E. Hendron ◽

S. Berneche ◽

...

Keyword(s):

Selectivity Filter ◽

K Channel ◽

Channel Selectivity

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Evidence for Sequential Ion-binding Loci along the Inner Pore of the IRK1 Inward-rectifier K+ Channel

The Journal of General Physiology ◽

10.1085/jgp.200509296 ◽

2005 ◽

Vol 126 (2) ◽

pp. 123-135 ◽

Cited By ~ 24

Author(s):

Hyeon-Gyu Shin ◽

Yanping Xu ◽

Zhe Lu

Keyword(s):

Voltage Dependence ◽

Inward Rectifier ◽

Selectivity Filter ◽

K Channel ◽

Ion Binding ◽

Side Chains ◽

Aromatic Side Chain ◽

Voltage Drops ◽

Inner Pore ◽

Aromatic Side Chains

Steep rectification in IRK1 (Kir2.1) inward-rectifier K+ channels reflects strong voltage dependence (valence of ∼5) of channel block by intracellular cationic blockers such as the polyamine spermine. The observed voltage dependence primarily results from displacement, by spermine, of up to five K+ ions across the narrow K+ selectivity filter, along which the transmembrane voltage drops steeply. Spermine first binds, with modest voltage dependence, at a shallow site where it encounters the innermost K+ ion and impedes conduction. From there, spermine can proceed to a deeper site, displacing several more K+ ions and thereby producing most of the observed voltage dependence. Since in the deeper blocked state the leading amine group of spermine reaches into the cavity region (internal to the selectivity filter) and interacts with residue D172, its trailing end is expected to be near M183. Here, we found that mutation M183A indeed affected the deeper blocked state, which supports the idea that spermine is located in the region lined by the M2 and not deep in the narrow K+ selectivity filter. As to the shallower site whose location has been unknown, we note that in the crystal structure of homologous GIRK1 (Kir3.1), four aromatic side chains of F255, one from each of the four subunits, constrict the intracellular end of the pore to ∼10 Å. For technical simplicity, we used tetraethylammonium (TEA) as an initial probe to test whether the corresponding residue in IRK1, F254, forms the shallower site. We found that replacing the aromatic side chain with an aliphatic one not only lowered TEA affinity of the shallower site ∼100-fold but also eliminated the associated voltage dependence and, furthermore, confirmed that similar effects occurred also for spermine. These results establish the evidence for physically separate, sequential ion-binding loci along the long inner pore of IRK1, and strongly suggest that the aromatic side chains of F254 underlie the likely innermost binding locus for both blocker and K+ ions in the cytoplasmic pore.

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S3h1-1 Molecular Determinants of Gating at the K^+ Channel Selectivity Filter, Probed by Protein Crystallography and Molecular Dynamics Simulations(S3-h1: "Structural Aspects of Channel and Transporter Proteins",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

Seibutsu Butsuri ◽

10.2142/biophys.46.s139_3 ◽

2006 ◽

Vol 46 (supplement2) ◽

pp. S139

Author(s):

Yanxiang Zhao ◽

Vish Jogini ◽

Eduardo Perozo ◽

Benoit Roux

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Protein Crystallography ◽

Selectivity Filter ◽

K Channel ◽

Meeting Program ◽

Channel Selectivity ◽

Molecular Determinants ◽

Dynamics Simulations ◽

Structural Aspects

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Two Stable, Conducting Conformations of the Selectivity Filter in Shaker K+ Channels

The Journal of General Physiology ◽

10.1085/jgp.200509251 ◽

2005 ◽

Vol 125 (6) ◽

pp. 619-629 ◽

Cited By ~ 7

Author(s):

Jill Thompson ◽

Ted Begenisich

Keyword(s):

Voltage Dependence ◽

Relative Proportion ◽

Selectivity Filter ◽

K Channel ◽

K Channels ◽

High Affinity ◽

K Concentration ◽

The One ◽

Low K ◽

Two Populations

We have examined the voltage dependence of external TEA block of Shaker K+ channels over a range of internal K+ concentrations from 2 to 135 mM. We found that the concentration dependence of external TEA block in low internal K+ solutions could not be described by a single TEA binding affinity. The deviation from a single TEA binding isotherm was increased at more depolarized membrane voltages. The data were well described by a two-component binding scheme representing two, relatively stable populations of conducting channels that differ in their affinity for external TEA. The relative proportion of these two populations was not much affected by membrane voltage but did depend on the internal K+ concentration. Low internal K+ promoted an increase in the fraction of channels with a low TEA affinity. The voltage dependence of the apparent high-affinity TEA binding constant depended on the internal K+ concentration, becoming almost voltage independent in 5 mM. The K+ sensitivity of these low- and high-affinity TEA states suggests that they may represent one- and two-ion occupancy states of the selectivity filter, consistent with recent crystallographic results from the bacterial KcsA K+ channel. We therefore analyzed these data in terms of such a model and found a large (almost 14-fold) difference between the intrinsic TEA affinity of the one-ion and two-ion modes. According to this analysis, the single ion in the one-ion mode (at 0 mV) prefers the inner end of the selectivity filter twofold more than the outer end. This distribution does not change with internal K+. The two ions in the two-ion mode prefer to occupy the inner end of the selectivity filter at low K+, but high internal K+ promotes increased occupancy of the outer sites. Our analysis further suggests that the four K+ sites in the selectivity filter are spaced between 20 and 25% of the membrane electric field.

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Structural basis for C-type inactivation in a Shaker family voltage gated K+ channel

10.1101/2021.10.01.462615 ◽

2021 ◽

Author(s):

Ravikumar Reddi ◽

Kimberly Matulef ◽

Erika A. Riederer ◽

Matthew R. Whorton ◽

Francis I. Valiyaveetil

Keyword(s):

Crystal Structure ◽

Binding Sites ◽

Structural Changes ◽

Selectivity Filter ◽

Structural Basis ◽

K Channel ◽

Ion Binding ◽

Channel Pore ◽

Voltage Gated ◽

Ion Binding Sites

AbstractC-type inactivation is a process by which ion flux through a voltage-gated K+ (Kv) channel is regulated at the selectivity filter. While prior studies have indicated that C-type inactivation involves structural changes at the selectivity filter, the nature of the changes have not been resolved. Here we report the crystal structure of the Kv1.2 channel in a C-type inactivated state. The structure shows that C-type inactivation involves changes in the selectivity filter that disrupt the outer two ion binding sites in the filter. The changes at the selectivity filter propagate to the extracellular mouth and the turret regions of the channel pore. The structural changes observed are consistent with the functional hallmarks of C-type inactivation. This study highlights the intricate interplay between K+ occupancy at the ion binding sites and the interactions of the selectivity filter in determining the balance between the conductive and the inactivated conformations of the filter.

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

The Journal of General Physiology ◽

10.1085/jgp.202012683 ◽

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.

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