Conformation Of Selectivity Filter Research Articles

Interactions between selectivity filter and pore helix control filter gating in the MthK channel.

K+ channel activity can be limited by C-type inactivation, which is likely initiated in part by dissociation of K+ ions from the selectivity filter and modulated by the side chains that surround it. While crystallographic and computational studies have linked inactivation to a "collapsed" selectivity filter conformation in the KcsA channel, the structural basis for selectivity filter gating in other K+ channels is less clear. Here, we combined electrophysiological recordings with molecular dynamics simulations, to study selectivity filter gating in the model potassium channel MthK and its V55E mutant (analogous to KcsA E71) in the pore-helix. We found that MthK V55E has a lower open probability than the WT channel, due to decreased stability of the open state, as well as a lower unitary conductance. Simulations account for both of these variables on the atomistic scale, showing that ion permeation in V55E is altered by two distinct orientations of the E55 side chain. In the "vertical" orientation, in which E55 forms a hydrogen bond with D64 (as in KcsA WT channels), the filter displays reduced conductance compared to MthK WT. In contrast, in the "horizontal" orientation, K+ conductance is closer to that of MthK WT; although selectivity filter stability is lowered, resulting in more frequent inactivation. Surprisingly, inactivation in MthK WT and V55E is associated with a widening of the selectivity filter, unlike what is observed for KcsA and reminisces recent structures of inactivated channels, suggesting a conserved inactivation pathway across the potassium channel family.

Rearrangement of a unique Kv1.3 selectivity filter conformation upon binding of a drug

We report two structures of the human voltage-gated potassium channel (Kv) Kv1.3 in immune cells alone (apo-Kv1.3) and bound to an immunomodulatory drug called dalazatide (dalazatide-Kv1.3). Both the apo-Kv1.3 and dalazatide-Kv1.3 structures are in an activated state based on their depolarized voltage sensor and open inner gate. In apo-Kv1.3, the aromatic residue in the signature sequence (Y447) adopts a position that diverges 11 Å from other K+ channels. The outer pore is significantly rearranged, causing widening of the selectivity filter and perturbation of ion binding within the filter. This conformation is stabilized by a network of intrasubunit hydrogen bonds. In dalazatide-Kv1.3, binding of dalazatide to the channel's outer vestibule narrows the selectivity filter, Y447 occupies a position seen in other K+ channels, and this conformation is stabilized by a network of intersubunit hydrogen bonds. These remarkable rearrangements in the selectivity filter underlie Kv1.3's transition into the drug-blocked state.

Mapping the Conformational States of the Outer Vestibule of Kcsa, a Prokaryote Potassium Channel, using the Long Chain Quaternary Ammonium Blocker, Tetraoctylammonium

KcsA is a homotetrameric prokaryotic potassium channel, homologous to its eukaryotic counterparts. In this study, we have used a KcsA mutant channel bearing a single tryptophan residue per monomer (W67 KcsA) to investigate the selectivity filter (SF) and pore-helix dynamics using fluorescence spectroscopy. Our steady-state and time-resolved fluorescence measurements indicate that the W67 residues undergo an efficient homo-FRET process among the four channel subunits. An analytical solution describing homo-FRET among a homo-tetramer in a square geometry allowed retrieving W67-W67 lateral distances (R) from the W67 KcsA fluorescence anisotropy decays. Comparison between the distances determined for the closed/conductive (pH 7.0, 200 mM K+, R=15.4 Å), closed/non-conductive (pH 7.0, 0.1 mM K+, R=16.5 Å) and open/inactivated (pH4.0, 200 mM K+, R=15.3 Å) of the detergent-solubilized W67 KcsA, ruled out the hypothesis that the inactivated state is structurally similar to its collapsed state. To further study the inactivated conformation, we added tetraoctylammonium (TOA+), which is a potassium channel blocker that binds to the internal cavity, beneath the SF. TOA+ allosterically triggers a structural rearrangement characterized by a decrease in the affinity for K+ at pH 7 and longer W67-W67 lateral distances, while the shorter alkyl chain salt tetrabutylammonium (TBA+) does not affect the SF conformation of KcsA. Such loss of affinity is similar to that seen for the open/inactivated state at pH 4. This allosteric modification is also linked to a decrease of the thermal stability of KcsA monitored by a thermal denaturation assay. These results reinforce the argument that long chain tetraalkyl ammonium salts, such as TOA+, stabilize an inactivated-like conformation of the SF, which has a lower affinity for K+ and a different conformation than the low-K+ (collapsed) structure.

Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA.

Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA+) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that serves as a model to its homologous eukaryotic counterparts. By the combined use of a thermal denaturation assay and the analysis of homo-Förster resonance energy transfer in a mutant channel containing a single tryptophan (W67) per subunit, we found that TOA+ binds the channel cavity with high affinity, either with the inner gate open or closed. Moreover, TOA+ bound at the cavity allosterically shifts the equilibrium of the channel’s selectivity filter conformation from conductive to an inactivated-like form. The inactivated TOA+–KcsA complex exhibits a loss in the affinity towards permeant K+ at pH 7.0, when the channel is in its closed state, but maintains the two sets of K+ binding sites and the W67–W67 intersubunit distances characteristic of the selectivity filter in the channel resting state. Thus, the TOA+–bound state differs clearly from the collapsed channel state described by X-ray crystallography and claimed to represent the inactivated form of KcsA.

Selectivity filter instability dominates the low intrinsic activity of the TWIK-1 K2P K+ channel

Two-pore domain K+ (K2P) channels have many important physiological functions. However, the functional properties of the TWIK-1 (K2P1.1/KCNK1) K2P channel remain poorly characterized because heterologous expression of this ion channel yields only very low levels of functional activity. Several underlying reasons have been proposed, including TWIK-1 retention in intracellular organelles, inhibition by posttranslational sumoylation, a hydrophobic barrier within the pore, and a low open probability of the selectivity filter (SF) gate. By evaluating these potential mechanisms, we found that the latter dominates the low intrinsic functional activity of TWIK-1. Investigating this further, we observed that the low activity of the SF gate appears to arise from the inefficiency of K+ in stabilizing an active (i.e. conductive) SF conformation. In contrast, other permeant ion species, such as Rb+, NH4+, and Cs+, strongly promoted a pH-dependent activated conformation. Furthermore, many K2P channels are activated by membrane depolarization via an SF-mediated gating mechanism, but we found here that only very strong nonphysiological depolarization produces voltage-dependent activation of heterologously expressed TWIK-1. Remarkably, we also observed that TWIK-1 Rb+ currents are potently inhibited by intracellular K+ (IC50 = 2.8 mm). We conclude that TWIK-1 displays unique SF gating properties among the family of K2P channels. In particular, the apparent instability of the conductive conformation of the TWIK-1 SF in the presence of K+ appears to dominate the low levels of intrinsic functional activity observed when the channel is expressed at the cell surface.

Electrostatic state of the cytoplasmic domain influences inactivation at the selectivity filter of the KcsA potassium channel

KcsA is a proton-activated K+ channel that is regulated at two gates: an activation gate located in the inner entrance of the pore and an inactivation gate at the selectivity filter. Previously, we revealed that the cytoplasmic domain (CPD) of KcsA senses proton and that electrostatic changes of the CPD influences the opening and closing of the activation gate. However, our previous studies did not reveal the effect of CPD on the inactivation gate because we used a non-inactivating mutant (E71A). In the present study, we used mutants that did not harbor the E71A mutation, and showed that the electrostatic state of the CPD influences the inactivation gate. Three novel CPD mutants were generated in which some negatively charged amino acids were replaced with neutral amino acids. These CPD mutants conducted K+, but showed various inactivation properties. Mutants carrying the D149N mutation showed high open probability and slow inactivation, whereas those without the D149N mutation showed low open probability and fast inactivation, similar to wild-type KcsA. In addition, mutants with D149N showed poor K+ selectivity, and permitted Na+ to flow. These results indicated that electrostatic changes in the CPD by D149N mutation triggered the loss of fast inactivation and changes in the conformation of selectivity filter. Additionally, the loss of fast inactivation induced by D149N was reversed by R153A mutation, suggesting that not only the electrostatic state of D149, but also that of R153 affects inactivation.

Converting the Depolarization-Activated Shaker KV Channel into a Hyperpolarization-Conducting Cation-Selective Channel by Two Pore Mutations

Substituting the pore residue W434 in the Shaker Kv channel by a phenylalanine (W434F) accelerates its C-type inactivation, which involves the collapse of the selectivity filter (SF). In Shaker-W434F this collapse precedes S6-gate opening upon activation and no ionic currents are recorded. However, a valine mutation for the residue T449 (T449V) slows inactivation and introducing this T449V substitution in the Shaker-W434F rescues ionic conduction. The human Shaker-type Kv1.2 channel has a valine at the homologous T449 position (V381). Interestingly, the Kv1.2-W366F mutant yielded ionic current (similar to the Shaker -W434F-T449V combination). Accordingly, substituting V381 in Kv1.2-W366F by a threonine resulted in a similar (non-conducting) phenotype as Shaker-W434F. Thus the rate of SF collapse can be controlled by specific combinations of pore residue mutations. Patch-clamp analysis of the ionic currents of Kv1.2-W366F and Shaker-W434F-T449V showed that the channels remained K+ selective. Thus, the SF of Shaker-W434F-T449V adopts a K+-selective conformation when the S6-gate opens. To study the SF conformation of Shaker-W434F when the S6-gate closes, we combined Shaker-W434F with the mutation P475D, that by itself prevents complete closing of the S6-gate. We expected to record ionic currents of the double Shaker-W434F-P475D mutant at hyperpolarized potentials when the S6-gate closes and the SF recovers from its collapsed inactivated state. Indeed, Shaker-W434F-P475D resulted in functional voltage-dependent channels that were conducting at hyperpolarized potentials and ceased conducting at depolarized potentials. Interestingly, this conductive state of Shaker-W434F-P475D was both Na+ and K+ permeable, i.e. the high K+ selectivity was lost. The mutant remained, however, sensitive to external TEA block. Thus, preventing full closure of the S6-gate appears to affect the recovery process of the SF such that it is trapped in a conformation conducting both Na+ and K+.

Allosteric coupling between proximal C-terminus and selectivity filter is facilitated by the movement of transmembrane segment 4 in TREK-2 channel.

TREK-2, a member of two-pore-domain potassium channel family, regulates cellular excitability in response to diverse stimuli. However, how such stimuli control channel function remains unclear. Here, by characterizing the responses of cytosolic proximal C-terminus deletant (ΔpCt) and transmembrane segment 4 (M4)-glycine hinge mutant (G312A) to 2-Aminoethoxydiphenyl borate (2-APB), an activator of TREK-2, we show that the transduction initiated from pCt domain is allosterically coupled with the conformation of selectivity filter (SF) via the movements of M4, without depending on the original status of SF. Moreover, ΔpCt and G312A also exhibited blunted responses to extracellular alkalization, a model to induce SF conformational transition. These results suggest that the coupling between pCt domain and SF is bidirectional, and M4 movements are involved in both processes. Further mechanistic exploration reveals that the function of Phe316, a residue close to the C-terminus of M4, is associated with such communications. However, unlike TREK-2, M4-hinge of TREK-1 only controls the transmission from pCt to SF, rather than SF conformational changes triggered by pHo changes. Together, our findings uncover the unique gating properties of TREK-2, and elucidate the mechanisms for how the extracellular and intracellular stimuli harness the pore gating allosterically.

The isoforms generated by alternative translation initiation adopt similar conformation in the selectivity filter in TREK-2.

TREK-2 (TWIK-related K(+) channel-2), a member of two-pore domain potassium (K2P) channel family, tunes cellular excitability via conducting leak or background currents. In TREK-2, the isoforms generated by alternative translation initiation (ATI) mechanism exhibit large divergence in unitary conductance, but similar in selectivity to K(+). Up to now, the structural basis for this similarity in ion selectivity is unknown. Here, we report that externally applied Ba(2+) inhibits the currents of TREK-2 in a concentration- and time-dependent manner. The blocking effect is blunted by elevated extracellular K(+) or mutation of S4 K(+) binding site, which suggests that the inhibitory mechanism of Ba(2+) is due to its competitive docking properties within the selectivity filter (SF). Next, we demonstrate that all the ATI isoforms exhibit analogous behaviors upon the application of Ba(2+) and alteration of extracellular pH (pHo), which acts on the outer position of the SF. These results strongly support the notion that all the ATI isoforms of TREK-2 possess resembled SF conformation in S4 site and the position defined by pHo, which implicates that neither the role of N-terminus (Nt) nor the unitary conductance is associated with SF conformation. Our findings might help to understand the detail gating mechanism of TREK-2 and K2P channels.

External Ba2+ Block of the Two-pore Domain Potassium Channel TREK-1 Defines Conformational Transition in Its Selectivity Filter

TREK-1 is a member of the two-pore domain potassium channel family that is known as a leak channel and plays a key role in many physiological and pathological processes. The conformational transition of the selectivity filter is considered as an effective strategy for potassium channels to control the course of potassium efflux. It is well known that TREK-1 is regulated by a large volume of extracellular and intracellular signals. However, until now, little was known about the selectivity filter gating mechanism of the channel. In this research, it was found that Ba(2+) blocked the TREK-1 channel in a concentration- and time-dependent manner. A mutagenesis analysis showed that overlapped binding of Ba(2+) at the assumed K(+) binding site 4 (S4) within the selectivity filter was responsible for the inhibitory effects on TREK-1. Then, Ba(2+) was used as a probe to explore the conformational transition in the selectivity filter of the channel. It was confirmed that collapsed conformations were induced by extracellular K(+)-free and acidification at the selectivity filters, leading to nonconductive to permeable ions. Further detailed characterization demonstrated that the two conformations presented different properties. Additionally, the N-terminal truncated isoform (ΔN41), a product derived from alternative translation initiation, was identified as a constitutively nonconductive variant. Together, these results illustrate the important role of selectivity filter gating in the regulation of TREK-1 by the extracellular K(+) and proton.

Conformational Changes in the Selectivity Filter of the Open-State KcsA Channel: An Energy Minimization Study

Potassium channels switch between closed and open conformations and selectively conduct K + ions. There are at least two gates. The TM2 bundle at the intracellular site is the primary gate of KcsA, and rearrangements at the selectivity filter (SF) act as the second gate. The SF blocks ion flow via an inactivation process similar to C-type inactivation of voltage-gated K + channels. We recently generated the open-state conformation of the KcsA channel. We found no major, possibly inactivating, structural changes in the SF associated with this massive inner-pore rearrangement, which suggests that the gates might act independently. Here we energy-minimize the open state of wild-type and mutant KcsA, validating in silico structures of energy-minimized SFs by comparison with crystallographic structures, and use these data to gain insight into how mutation, ion depletion, and K + to Na + substitution influence SF conformation. Both E71 or D80 protonations/mutations and the presence/absence of protein-buried water molecule(s) modify the H-bonding network stabilizing the P-loops, spawning numerous SF conformations. We find that the inactivated state corresponds to conformations with a partially unoccupied or an entirely empty SF. These structures, involving modifications in all four P-loops, are stabilized by H-bonds between amide H and carbonyl O atoms from adjacent P-loops, which block ion passage. The inner portions of the P-loops are more rigid than the outer parts. Changes are localized to the outer binding sites, with innermost site S4 persisting in the inactivated state. Strong binding by Na + locally contracts the SF around Na +, releasing ligands that do not participate in Na + coordination, and occluding the permeation pathway. K + selectivity primarily appears to arise from the inability of the SF to completely dehydrate Na + ions due to basic structural differences between liquid water and the “quasi-liquid” SF matrix.