Halting KcsA'S C-Type Inactivation Gating by Controlling Water Diffusion Behind the Channel'S Selectivity Filter

The amount of ionic current flowing through K+ channels is determined by the interplay between two separate time-dependent processes: activation and inactivation gating. Activation is concerned with the stimulus-dependent opening of the main intracellular gate, whereas inactivation is a spontaneous conformational transition of the selectivity filter toward a nonconductive state occurring on a variety of timescales. A recent analysis of multiple x-ray structures of open and partially open KcsA channels revealed the mechanism by which movements of the inner activation gate, formed by the inner helices from the four subunits of the pore domain, bias the conformational changes at the selectivity filter toward a nonconductive inactivated state. This analysis highlighted the important role of Phe103, a residue located along the inner helix, near the hinge position associated with the opening of the intracellular gate. In the present study, we use free energy perturbation molecular dynamics simulations (FEP/MD) to quantitatively elucidate the thermodynamic basis for the coupling between the intracellular gate and the selectivity filter. The results of the FEP/MD calculations are in good agreement with experiments, and further analysis of the repulsive, van der Waals dispersive, and electrostatic free energy contributions reveals that the energetic basis underlying the absence of inactivation in the F103A mutation in KcsA is the absence of the unfavorable steric interaction occurring with the large Ile100 side chain in a neighboring subunit when the intracellular gate is open and the selectivity filter is in a conductive conformation. Macroscopic current analysis shows that the I100A mutant indeed relieves inactivation in KcsA, but to a lesser extent than the F103A mutant.

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The gating cycle of a K+ channel at atomic resolution

eLife ◽

10.7554/elife.28032 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 39

Author(s):

Luis G Cuello ◽

D Marien Cortes ◽

Eduardo Perozo

Keyword(s):

Conformational Changes ◽

Selectivity Filter ◽

Atomic Resolution ◽

K Channel ◽

Allosteric Interactions ◽

Inactivation Gating ◽

Pore Domain ◽

Fine Tune ◽

Conductive State

C-type inactivation in potassium channels helps fine-tune long-term channel activity through conformational changes at the selectivity filter. Here, through the use of cross-linked constitutively open constructs, we determined the structures of KcsA’s mutants that stabilize the selectivity filter in its conductive (E71A, at 2.25 Å) and deep C-type inactivated (Y82A at 2.4 Å) conformations. These structural snapshots represent KcsA’s transient open-conductive (O/O) and the stable open deep C-type inactivated states (O/I), respectively. The present structures provide an unprecedented view of the selectivity filter backbone in its collapsed deep C-type inactivated conformation, highlighting the close interactions with structural waters and the local allosteric interactions that couple activation and inactivation gating. Together with the structures associated with the closed-inactivated state (C/I) and in the well-known closed conductive state (C/O), this work recapitulates, at atomic resolution, the key conformational changes of a potassium channel pore domain as it progresses along its gating cycle.

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Gating of two-pore domain K+ channels by extracellular pH

Biochemical Society Transactions ◽

10.1042/bst0340899 ◽

2006 ◽

Vol 34 (5) ◽

pp. 899-902 ◽

Cited By ~ 13

Author(s):

M.I. Niemeyer ◽

F.D. González-Nilo ◽

L. Zúñiga ◽

W. González ◽

L.P. Cid ◽

...

Keyword(s):

Extracellular Ph ◽

Selectivity Filter ◽

Open Probability ◽

K Channels ◽

Task Channels ◽

Pore Mouth ◽

Inactivation Gating ◽

Opening And Closing ◽

Pore Domain ◽

And Task

Potassium channels have a conserved selectivity filter that is important in determining which ions are conducted and at what rate. Although K+ channels of different conductance characteristics are known, they differ more widely in the way their opening and closing, the gating, is governed. TASK and TALK subfamily proteins are two-pore region KCNK K+ channels gated open by extracellular pH. We discuss the mechanism for this gating in terms of electrostatic effects on the pore changing the occupancy and open probability of the channels in a way reminiscent of C-type inactivation gating at the selectivity filter. Essential to this proposed mechanism is the replacement of two highly conserved aspartate residues at the pore mouth by asparagine or histidine residues in the TALK and TASK channels.

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A unique mechanism of inactivation gating of the Kv channel family member Kv7.1 and its modulation by PIP2 and calmodulin

Science Advances ◽

10.1126/sciadv.abd6922 ◽

2020 ◽

Vol 6 (51) ◽

pp. eabd6922

Author(s):

Maya Lipinsky ◽

William Sam Tobelaim ◽

Asher Peretz ◽

Luba Simhaev ◽

Adva Yeheskel ◽

...

Keyword(s):

Family Member ◽

Selectivity Filter ◽

Wild Type ◽

Voltage Gated ◽

Kv Channel ◽

Kv Channels ◽

Dependent Inactivation ◽

Voltage Dependent ◽

Inactivation Gating ◽

Unique Mechanism

Inactivation of voltage-gated K+ (Kv) channels mostly occurs by fast N-type or/and slow C-type mechanisms. Here, we characterized a unique mechanism of inactivation gating comprising two inactivation states in a member of the Kv channel superfamily, Kv7.1. Removal of external Ca2+ in wild-type Kv7.1 channels produced a large, voltage-dependent inactivation, which differed from N- or C-type mechanisms. Glu295 and Asp317 located, respectively, in the turret and pore entrance are involved in Ca2+ coordination, allowing Asp317 to form H-bonding with the pore helix Trp304, which stabilizes the selectivity filter and prevents inactivation. Phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+-calmodulin prevented Kv7.1 inactivation triggered by Ca2+-free external solutions, where Ser182 at the S2-S3 linker relays the calmodulin signal from its inner boundary to the external pore to allow proper channel conduction. Thus, we revealed a unique mechanism of inactivation gating in Kv7.1, exquisitely controlled by external Ca2+ and allosterically coupled by internal PIP2 and Ca2+-calmodulin.

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Rapid constriction of the selectivity filter underlies C-type inactivation in the KcsA potassium channel

The Journal of General Physiology ◽

10.1085/jgp.201812082 ◽

2018 ◽

Vol 150 (10) ◽

pp. 1408-1420 ◽

Cited By ~ 27

Author(s):

Jing Li ◽

Jared Ostmeyer ◽

Luis G. Cuello ◽

Eduardo Perozo ◽

Benoît Roux

Keyword(s):

Ionic Conduction ◽

Md Simulations ◽

Umbrella Sampling ◽

Potential Of Mean Force ◽

Selectivity Filter ◽

Truncated Form ◽

Terminal Domain ◽

Activation Gate ◽

Electrophysiological Measurements ◽

Inactivation Gating

C-type inactivation is a time-dependent process observed in many K+ channels whereby prolonged activation by an external stimulus leads to a reduction in ionic conduction. While C-type inactivation is thought to be a result of a constriction of the selectivity filter, the local dynamics of the process remain elusive. Here, we use molecular dynamics (MD) simulations of the KcsA channel to elucidate the nature of kinetically delayed activation/inactivation gating coupling. Microsecond-scale MD simulations based on the truncated form of the KcsA channel (C-terminal domain deleted) provide a first glimpse of the onset of C-type inactivation. We observe over multiple trajectories that the selectivity filter consistently undergoes a spontaneous and rapid (within 1–2 µs) transition to a constricted conformation when the intracellular activation gate is fully open, but remains in the conductive conformation when the activation gate is closed or partially open. Multidimensional umbrella sampling potential of mean force calculations and nonequilibrium voltage-driven simulations further confirm these observations. Electrophysiological measurements show that the truncated form of the KcsA channel inactivates faster and greater than full-length KcsA, which is consistent with truncated KcsA opening to a greater degree because of the absence of the C-terminal domain restraint. Together, these results imply that the observed kinetics underlying activation/inactivation gating reflect a rapid conductive-to-constricted transition of the selectivity filter that is allosterically controlled by the slow opening of the intracellular gate.

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Inverted allosteric coupling between activation and inactivation gates in K+ channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1800559115 ◽

2018 ◽

Vol 115 (21) ◽

pp. 5426-5431 ◽

Cited By ~ 21

Author(s):

Alain J. Labro ◽

D. Marien Cortes ◽

Cholpon Tilegenova ◽

Luis G. Cuello

Keyword(s):

Selectivity Filter ◽

K Channels ◽

Allosteric Coupling ◽

Allosteric Communication ◽

Crystallographic Study ◽

State Dependent ◽

Activation Gate ◽

Activation Gating ◽

Inactivation Gating ◽

Signature Sequence

The selectivity filter and the activation gate in potassium channels are functionally and structurally coupled. An allosteric coupling underlies C-type inactivation coupled to activation gating in this ion-channel family (i.e., opening of the activation gate triggers the collapse of the channel’s selectivity filter). We have identified the second Threonine residue within the TTVGYGD signature sequence of K+ channels as a crucial residue for this allosteric communication. A Threonine to Alanine substitution at this position was studied in three representative members of the K+-channel family. Interestingly, all of the mutant channels exhibited lack of C-type inactivation gating and an inversion of their allosteric coupling (i.e., closing of the activation gate collapses the channel’s selectivity filter). A state-dependent crystallographic study of KcsA-T75A proves that, on activation, the selectivity filter transitions from a nonconductive and deep C-type inactivated conformation to a conductive one. Finally, we provide a crystallographic demonstration that closed-state inactivation can be achieved by the structural collapse of the channel’s selectivity filter.

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Voltage-Dependent Inactivation Gating at the Selectivity Filter of the MthK K+ channel

Biophysical Journal ◽

10.1016/j.bpj.2010.12.3364 ◽

2011 ◽

Vol 100 (3) ◽

pp. 582a

Author(s):

Andrew S. Thomson ◽

Brad S. Rothberg

Keyword(s):

Selectivity Filter ◽

K Channel ◽

Dependent Inactivation ◽

Voltage Dependent ◽

Inactivation Gating

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Voltage-dependent inactivation gating at the selectivity filter of the MthK K+ channel

The Journal of General Physiology ◽

10.1085/jgp.201010507 ◽

2010 ◽

Vol 136 (5) ◽

pp. 569-579 ◽

Cited By ~ 25

Author(s):

Andrew S. Thomson ◽

Brad S. Rothberg

Keyword(s):

Voltage Sensor ◽

Selectivity Filter ◽

K Channel ◽

K Channels ◽

Dependent Inactivation ◽

Wide Range ◽

Voltage Dependent ◽

Inactivation Gating ◽

External K ◽

Conductive State

Voltage-dependent K+ channels can undergo a gating process known as C-type inactivation, which involves entry into a nonconducting state through conformational changes near the channel’s selectivity filter. C-type inactivation may involve movements of transmembrane voltage sensor domains, although the mechanisms underlying this form of inactivation may be heterogeneous and are often unclear. Here, we report on a form of voltage-dependent inactivation gating observed in MthK, a prokaryotic K+ channel that lacks a canonical voltage sensor and may thus provide a reduced system to inform on mechanism. In single-channel recordings, we observe that Po decreases with depolarization, with a half-maximal voltage of 96 ± 3 mV. This gating is kinetically distinct from blockade by internal Ca2+ or Ba2+, suggesting that it may arise from an intrinsic inactivation mechanism. Inactivation gating was shifted toward more positive voltages by increasing external [K+] (47 mV per 10-fold increase in [K+]), suggesting that K+ binding at the extracellular side of the channel stabilizes the open-conductive state. The open-conductive state was stabilized by other external cations, and selectivity of the stabilizing site followed the sequence: K+ ≈ Rb+ > Cs+ > Na+ > Li+ ≈ NMG+. Selectivity of the stabilizing site is weaker than that of sites that determine permeability of these ions, suggesting that the site may lie toward the external end of the MthK selectivity filter. We could describe MthK gating over a wide range of positive voltages and external [K+] using kinetic schemes in which the open-conductive state is stabilized by K+ binding to a site that is not deep within the electric field, with the voltage dependence of inactivation arising from both voltage-dependent K+ dissociation and transitions between nonconducting (inactivated) states. These results provide a quantitative working hypothesis for voltage-dependent, K+-sensitive inactivation gating, a property that may be common to other K+ channels.

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