Shaker-IR K Channel Gating in Heavy Water: Role of Structural Water Molecules in Inactivation

C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K+ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K+ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.

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Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel

Shen

Rohaim

et al. 2021

Journal of General Physiology

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Cm28, a scorpion toxin having a unique primary structure, inhibits KV1.2 and KV1.3 with high affinity

Naseem

Carcamo-Noriega

Beltrán-Vidal

et al. 2022

Journal of General Physiology

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The Cm28 in the venom of Centruroides margaritatus is a short peptide consisting of 27 amino acid residues with a mol wt of 2,820 D. Cm28 has <40% similarity with other known α-KTx from scorpions and lacks the typical functional dyad (lysine–tyrosine) required to block KV channels. However, its unique sequence contains the three disulfide-bond traits of the α-KTx scorpion toxin family. We propose that Cm28 is the first example of a new subfamily of α-KTxs, registered with the systematic number α-KTx32.1. Cm28 inhibited voltage-gated K+ channels KV1.2 and KV1.3 with Kd values of 0.96 and 1.3 nM, respectively. There was no significant shift in the conductance–voltage (G-V) relationship for any of the channels in the presence of toxin. Toxin binding kinetics showed that the association and dissociation rates are consistent with a bimolecular interaction between the peptide and the channel. Based on these, we conclude that Cm28 is not a gating modifier but rather a pore blocker. In a selectivity assay, Cm28 at 150 nM concentration (>100× Kd value for KV1.3) did not inhibit KV1.5, KV11.1, KCa1.1, and KCa3.1 K+ channels; NaV1.5 and NaV1.4 Na+ channels; or the hHV1 H+ channel but blocked ∼27% of the KV1.1 current. In a biological functional assay, Cm28 strongly inhibited the expression of the activation markers interleukin-2 receptor and CD40 ligand in anti-CD3–activated human CD4+ effector memory T lymphocytes. Cm28, due to its unique structure, may serve as a template for the generation of novel peptides targeting KV1.3 in autoimmune diseases.

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Molecular rearrangements in S6 during slow inactivation in Shaker-IR potassium channels

Szanto

Papp

Zakany

et al. 2023

Journal of General Physiology

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Voltage-gated K+ channels have distinct gates that regulate ion flux: the activation gate (A-gate) formed by the bundle crossing of the S6 transmembrane helices and the slow inactivation gate in the selectivity filter. These two gates are bidirectionally coupled. If coupling involves the rearrangement of the S6 transmembrane segment, then we predict state-dependent changes in the accessibility of S6 residues from the water-filled cavity of the channel with gating. To test this, we engineered cysteines, one at a time, at S6 positions A471, L472, and P473 in a T449A Shaker-IR background and determined the accessibility of these cysteines to cysteine-modifying reagents MTSET and MTSEA applied to the cytosolic surface of inside-out patches. We found that neither reagent modified either of the cysteines in the closed or the open state of the channels. On the contrary, A471C and P473C, but not L472C, were modified by MTSEA, but not by MTSET, if applied to inactivated channels with open A-gate (OI state). Our results, combined with earlier studies reporting reduced accessibility of residues I470C and V474C in the inactivated state, strongly suggest that the coupling between the A-gate and the slow inactivation gate is mediated by rearrangements in the S6 segment. The S6 rearrangements are consistent with a rigid rod-like rotation of S6 around its longitudinal axis upon inactivation. S6 rotation and changes in its environment are concomitant events in slow inactivation of Shaker KV channels.

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Shaker-IR K Channel Gating in Heavy Water: Role of Structural Water Molecules in Inactivation

Cited by 4 publications

References 79 publications

Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel

Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel

Cm28, a scorpion toxin having a unique primary structure, inhibits KV1.2 and KV1.3 with high affinity

Molecular rearrangements in S6 during slow inactivation in Shaker-IR potassium channels

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