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

2005 ◽  
Vol 126 (2) ◽  
pp. 123-135 ◽  
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.

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

2005 ◽  
Vol 125 (4) ◽  
pp. 413-426 ◽  
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.

scholarly journals Na+ Block and Permeation in a K+ Channel of Known Structure

2002 ◽  
Vol 120 (3) ◽  
pp. 323-335 ◽  
Author(s):  
Crina M. Nimigean ◽  
Christopher Miller
Keyword(s):  
High Voltage ◽  
Voltage Dependence ◽  
Low Voltage ◽  
Selectivity Filter ◽  
K Channel ◽  
Voltage Dependent

The effects of intracellular Na+ were studied on K+ and Rb+ currents through single KcsA channels. At low voltage, Na+ produces voltage-dependent block, which becomes relieved at high voltage by a “punchthrough” mechanism representing Na+ escaping from its blocking site through the selectivity filter. The Na+ blocking site is located in the wide, hydrated vestibule, and it displays unexpected selectivity for K+ and Rb+ against Na+. The voltage dependence of Na+ block reflects coordinated movements of the blocker with permeant ions in the selectivity filter.

scholarly journals Coupled Ion Movement Underlies Rectification in an Inward-Rectifier K+ Channel

1998 ◽  
Vol 112 (2) ◽  
pp. 211-221 ◽  
Author(s):  
Maria Spassova ◽  
Zhe Lu
Keyword(s):  
Voltage Dependence ◽  
Inward Rectifier ◽  
Membrane Voltage ◽  
Equilibrium Potential ◽  
Inward Rectification ◽  
K Channel ◽  
Channel Blockade ◽  
Electrical Field ◽  
K Current ◽  
Internal Pore

We studied block of the internal pore of the ROMK1 inward-rectifier K+ channel by Mg2+ and five quaternary ammoniums (tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, and tetrapentylammonium). The apparent affinity of these blockers varied as a function of membrane voltage. As a consequence, the channel conducted K+ current more efficiently in the inward than the outward direction; i.e., inward rectification. Although the size of some monovalent quaternary ammoniums is rather large, the zδ values (which measure voltage dependence of their binding to the pore) were near unity in symmetric 100 mM K+. Furthermore, we observed that not only the apparent affinities of the blockers themselves, but also their dependence on membrane voltage (or zδ), varied as a function of the concentration of extracellular K+. These results suggest that there is energetic coupling between the binding of blocking and permeating (K+) ions, and that the voltage dependence of channel blockade results, at least in part, from the movement of K+ ions in the electrical field. A further quantitative analysis of the results explains why the complex phenomenon of inward rectification depends on both membrane voltage and the equilibrium potential for K+.

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

2005 ◽  
Vol 125 (6) ◽  
pp. 619-629 ◽  
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.

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

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.

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

2015 ◽  
Vol 112 (49) ◽  
pp. 15096-15100 ◽  
Author(s):  
Shian Liu ◽  
Paul J. Focke ◽  
Kimberly Matulef ◽  
Xuelin Bian ◽  
Pierre Moënne-Loccoz ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Selectivity Filter ◽  
Titration Calorimetry ◽  
K Channel ◽  
Ion Binding ◽  
Binding Properties ◽  
Channel Opening ◽  
Channel Selectivity ◽  
Inactivation Process ◽  
Low K

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.

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 Interactions of external K+ and internal blockers in a weak inward-rectifier K+ channel

2012 ◽  
Vol 140 (5) ◽  
pp. 529-540 ◽  
Author(s):  
Lei Yang ◽  
Johan Edvinsson ◽  
Lawrence G. Palmer
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Inward Rectifier ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
C Terminus ◽  
Single Channel Currents ◽  
A Site ◽  
Channel Currents ◽  
External K

We investigated the effects of changing extracellular K+ concentrations on block of the weak inward-rectifier K+ channel Kir1.1b (ROMK2) by the three intracellular cations Mg2+, Na+, and TEA+. Single-channel currents were monitored in inside-out patches made from Xenopus laevis oocytes expressing the channels. With 110 mM K+ in the inside (cytoplasmic) solution and 11 mM K+ in the outside (extracellular) solution, these three cations blocked K+ currents with a range of apparent affinities (Ki (0) = 1.6 mM for Mg2+, 160 mM for Na+, and 1.8 mM for TEA+) but with similar voltage dependence (zδ = 0.58 for Mg2+, 0.71 for Na+, and 0.61 for TEA+) despite having different valences. When external K+ was increased to 110 mM, the apparent affinity of all three blockers was decreased approximately threefold with no significant change in the voltage dependence of block. The possibility that the transmembrane cavity is the site of block was explored by making mutations at the N152 residue, a position previously shown to affect rectification in Kir channels. N152D increased the affinity for block by Mg2+ but not for Na+ or TEA+. In contrast, the N152Y mutation increased the affinity for block by TEA+ but not for Na+ or Mg2+. Replacing the C terminus of the channel with that of the strong inward-rectifier Kir2.1 increased the affinity of block by Mg2+ but had a small effect on that by Na+. TEA+ block was enhanced and had a larger voltage dependence. We used an eight-state kinetic model to simulate these results. The effects of voltage and external K+ could be explained by a model in which the blockers occupy a site, presumably in the transmembrane cavity, at a position that is largely unaffected by changes in the electric field. The effects of voltage and extracellular K+ are explained by shifts in the occupancy of sites within the selectivity filter by K+ ions.

scholarly journals Locale and chemistry of spermine binding in the archetypal inward rectifier Kir2.1

2010 ◽  
Vol 135 (5) ◽  
pp. 495-508 ◽  
Author(s):  
Harley T. Kurata ◽  
Emily A. Zhu ◽  
Colin G. Nichols
Keyword(s):  
Binding Site ◽  
Voltage Dependence ◽  
Inward Rectifier ◽  
Selectivity Filter ◽  
Inward Rectification ◽  
Before And After ◽  
Voltage Dependent ◽  
Inwardly Rectifying ◽  
Kinetics Of

Polyamine block of inwardly rectifying potassium (Kir) channels underlies their steep voltage dependence observed in vivo. We have examined the potency, voltage dependence, and kinetics of spermine block in dimeric Kir2.1 constructs containing one nonreactive subunit and one cysteine-substituted subunit before and after modification by methanethiosulfonate (MTS) reagents. At position 169C (between the D172 “rectification controller” and the selectivity filter), modification by either 2-aminoethyl MTS (MTSEA) or 2-(trimethylammonium)ethyl MTS (MTSET) reduced the potency and voltage dependence of spermine block, consistent with this position overlapping the spermine binding site. At position 176C (between D172 and the M2 helix bundle crossing), modification by MTSEA also weakened spermine block. In contrast, MTSET modification of 176C dramatically slowed the kinetics of spermine unblock, with almost no effect on potency or voltage dependence. The data are consistent with MTSET modification of 176C introducing a localized barrier in the inner cavity, resulting in slower spermine entry into and exit from a “deep” binding site (likely between the D172 rectification controller and the selectivity filter), but leaving the spermine binding site mostly unaffected. These findings constrain the location of deep spermine binding that underlies steeply voltage-dependent block, and further suggest important chemical details of high affinity binding of spermine in Kir2.1 channels—the archetypal model of strong inward rectification.

Sign in / Sign up

Export Citation Format

Share Document