Inactivation Gating Research Articles

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

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.

The common features of tetrameric ion channels and the role of electrostatic interactions

The three tetrameric voltage-gated K+, Na+, and Ca2+ ion channels share a high number of common structural features. Moreover, they share a common charge distribution over the corresponding molecular components. These common features point to an energetically favored pathway of channel opening. This pathway starts from an ion channel conformation consisting of the activation gate closed and the inactivation gate (i.e., the selectivity filter) open. As a consequence of a depolarizing pulse, the stepwise outward movement of the four S4 helices triggers a series of interlinked electrostatic attractive interactions leading to complete opening of the ion channels. The assumption of a stepwise outward movement of the S4 helices is supported not only by the possibility of accounting for the salient kinetic features of these ion channels with a minimum of two, or at most three, free parameters, but also by a similar experimental stepwise movement of the gating charge.

Variable patterns of mutation density among NaV1.1, NaV1.2 and NaV1.6 point to channel-specific functional differences associated with childhood epilepsy.

Variants implicated in childhood epilepsy have been identified in all four voltage-gated sodium channels that initiate action potentials in the central nervous system. Previous research has focused on the functional effects of particular variants within the most studied of these channels (NaV1.1, NaV1.2 and NaV1.6); however, there have been few comparative studies across channels to infer the impact of mutations in patients with epilepsy. Here we compare patterns of variation in patient and public databases to test the hypothesis that regions of known functional significance within voltage-gated sodium (NaV) channels have an increased burden of deleterious variants. We assessed mutational burden in different regions of the Nav channels by (1) performing Fisher exact tests on odds ratios to infer excess variants in domains, segments, and loops of each channel in patient databases versus public "control" databases, and (2) comparing the cumulative distribution of variant sites along DNA sequences of each gene in patient and public databases (i.e., independent of protein structure). Patient variant density was concordant among channels in regions known to play a role in channel function, with statistically significant higher patient variant density in S4-S6 and DIII-DIV and an excess of public variants in SI-S3, DI-DII, DII-DIII. On the other hand, channel-specific patterns of patient burden were found in the NaV1.6 inactivation gate and NaV1.1 S5-S6 linkers, while NaV1.2 and NaV1.6 S4-S5 linkers and S5 segments shared patient variant patterns that contrasted with those in NaV1.1. These different patterns may reflect different roles played by the NaV1.6 inactivation gate in action potential propagation, and by NaV1.1 S5-S6 linkers in loss of function and haploinsufficiency. Interestingly, NaV1.2 and NaV1.6 both lack amino acid substitutions over significantly long stretches in both the patient and public databases suggesting that new mutations in these regions may cause embryonic lethality or a non-epileptic disease phenotype.

Modulation of Kv4.2/KChIP3 interaction by the ceroid lipofuscinosis neuronal 3 protein CLN3

Voltage-gated potassium (Kv) channels of the Kv4 subfamily associate with Kv channel-interacting proteins (KChIPs), which leads to enhanced surface expression and shapes the inactivation gating of these channels. KChIP3 has been reported to also interact with the late endosomal/lysosomal membrane glycoprotein CLN3 (ceroid lipofuscinosis neuronal 3), which is modified because of gene mutation in juvenile neuronal ceroid lipofuscinosis (JNCL). The present study was undertaken to find out whether and how CLN3, by its interaction with KChIP3, may indirectly modulate Kv4.2 channel expression and function. To this end, we expressed KChIP3 and CLN3, either individually or simultaneously, together with Kv4.2 in HEK 293 cells. We performed co-immunoprecipitation experiments and found a lower amount of KChIP3 bound to Kv4.2 in the presence of CLN3. In whole-cell patch-clamp experiments, we examined the effects of CLN3 co-expression on the KChIP3-mediated modulation of Kv4.2 channels. Simultaneous co-expression of CLN3 and KChIP3 with Kv4.2 resulted in a suppression of the typical KChIP3-mediated modulation; i.e. we observed less increase in current density, less slowing of macroscopic current decay, less acceleration of recovery from inactivation, and a less positively shifted voltage dependence of steady-state inactivation. The suppression of the KChIP3-mediated modulation of Kv4.2 channels was weaker for the JNCL-related missense mutant CLN3R334C and for a JNCL-related C-terminal deletion mutant (CLN3ΔC). Our data support the notion that CLN3 is involved in Kv4.2/KChIP3 somatodendritic A-type channel formation, trafficking, and function, a feature that may be lost in JNCL.

Deterministic model of Cav3.1 Ca2+ channel and a proposed sequence of its conformations

A family of current–time curves of T-type Cav3.1 Ca2+ channels available in the literature is simulated by a kinetic model differing from that used for the interpretation of all salient features of Na+ and Shaker K+ channels by the insertion of a multiplying factor expressing the difference between the working potential ϕ and the reversal potential ϕr. This deterministic model is also used to simulate experimental curves taken from the literature for steady-state ‘fast inactivation’ and for a gradual passage from fast to ‘slow inactivation’. A depolarizing pulse induces fast or slow inactivation depending on whether it lasts 100–500 ms or about 1 min, and is believed to cause a collapse of the central pore near the selectivity filter (SF). A number of features of fast and slow inactivation of Cav3.1 Ca2+ channels are qualitatively interpreted on the basis of a sequence of conformational states. Briefly, the conformation responsible for ‘fast inactivation’ is assumed to have the activation gate open and the inactivation gate (i.e., the SF) inactive. Immediately after a depolarizing pulse, this conformation is inactive and requires a sufficiently long rest time at a far negative holding potential to recover from inactivation. ‘Slow inactivation’ is ascribed to a different conformation with the activation gate closed and the SF inactive.

E1784K, the most common Brugada syndrome and long-QT syndrome type 3 mutant, disrupts sodium channel inactivation through two separate mechanisms.

Inheritable and de novo variants in the cardiac voltage-gated sodium channel, Nav1.5, are responsible for both long-QT syndrome type 3 (LQT3) and Brugada syndrome type 1 (BrS1). Interestingly, a subset of Nav1.5 variants can cause both LQT3 and BrS1. Many of these variants are found in channel structures that form the channel fast inactivation machinery, altering the rate, voltage dependence, and completeness of the fast inactivation process. We used a series of mutants at position 1784 to show that the most common inheritable Nav1.5 variant, E1784K, alters fast inactivation through two separable mechanisms: (1) a charge-dependent interaction that increases the noninactivating current characteristic of E1784K; and (2) a hyperpolarized voltage dependence and accelerated rate of fast inactivation that decreases the peak sodium current. Using a homology model built on the NavPaS structure, we find that the charge-dependent interaction is between E1784 and K1493 in the DIII-DIV linker of the channel, five residues downstream of the putative inactivation gate. This interaction can be disrupted by a positive charge at position 1784 and rescued with the K1493E/E1784K double mutant that abolishes the noninactivating current. However, the double mutant does not restore either the voltage dependence or rates of fast inactivation. Conversely, a mutant at the bottom of DIVS4, K1641D, causes a hyperpolarizing shift in the voltage dependence of fast inactivation and accelerates the rate of fast inactivation without causing an increase in noninactivating current. These findings provide novel mechanistic insights into how the most common inheritable arrhythmogenic mixed syndrome variant, E1784K, simultaneously decreases transient sodium currents and increases noninactivating currents, leading to both BrS1 and LQT3.

Flow and shortcuts along the Shaker Kv channel slow inactivation gating cycle.

Closing the cycle of Kv channel slow inactivation gating.

The Selectivity Filter Is Involved in the U-Type Inactivation Process of Kv2.1 and Kv3.1 Channels

Voltage-gated potassium (Kv) channels display several types of inactivation processes, including N-, C-, and U-types. C-type inactivation is attributed to a nonconductive conformation of the selectivity filter (SF). It has been proposed that the activation gate and the channel’s SF are allosterically coupled because the conformational changes of the former affect the structure of the latter and vice versa. The second threonine of the SF signature sequence (e.g., TTVGYG) has been proven to be essential for this allosteric coupling. To further study the role of the SF in U-type inactivation, we substituted the second threonine of the TTVGYG sequence by an alanine in the hKv2.1 and hKv3.1 channels, which are known to display U-type inactivation. Both hKv2.1-T377A and hKv3.1-T400A yielded channels that were resistant to inactivation, and as a result, they displayed noninactivating currents upon channel opening; i.e., hKv2.1-T377A and hKv3.1-T400A remained fully conductive upon prolonged moderate depolarizations, whereas in wild-type hKv2.1 and hKv3.1, the current amplitude typically reduces because of U-type inactivation. Interestingly, increasing the extracellular K+ concentration increased the macroscopic current amplitude of both hKv2.1-T377A and hKv3.1-T400A, which is similar to the response of the homologous T to A mutation in Shaker and hKv1.5 channels that display C-type inactivation. Our data support an important role for the second threonine of the SF signature sequence in the U-type inactivation gating of hKv2.1 and hKv3.1.

Revisiting NaV Channel Inactivation: The Role of Fibroblast Growth Factor Homologous Factor (FHF)

Cardiac sodium channel (NaV1.5) activation underlies the initiation and propagation of the heartbeat and is regulated by numerous accessory proteins. NaV1.5 comprises 4 homologous domains (DI-DIV), each consisting of 6 transmembrane segments (S1-S6) constituting the voltage sensing domain (VSD; S1-S4) and the channel pore (S5-S6). FHF, or intracellular fibroblast growth factors (iFGF), were found to regulate NaV1.5 kinetics through C-terminal binding near Calmodulin (CaM), but the mechanism remains unclear. To probe this mechanism, we observed the regulation of Nav1.5 VSDs by the cardiac-predominant FHF1B (iFGF12B), and the alternative FHF1A (iFGF12A) splice variant, using voltage-clamp fluorometry (VCF) in the cut-open configuration for individual VSD monitoring. Voltage clamp and patch clamp were also employed for electrophysiological studies in Xenopus Oocytes and CHO cells. We found that FHF1A and FHF1B regulate NaV1.5 inactivation by causing depolarizing shifts in the steady-state inactivation (SSI) curve and the DIII-VSD activation. Disruption of the CaM binding motif (IQ/AA) did not alter the SSI shift, suggesting CaM-independent FHF modulation. However, mutation of the inactivation gate (IFM motif) within DIII-IV linker (F1486Q) negates the FHF's effects on DIII-VSD and SSI. We conclude that FHF1s regulate NaV1.5 inactivation through an inactivation gate, independent of CaM. Furthermore, the A-type FHF counteracts impaired inactivation of F1486Q-NaV1.5 and reduces the late sodium current. A peptide bearing the first 21 amino acids of FHF1A's N-terminus is insufficient to reduce the late sodium current, implying different mechanism than pore blocking. Similar inactivation rescue by FHF1A was observed in long QT syndrome-linked mutations (ΔKPQ, ΔK1500 and F1473S), without affecting the shifts in SSI nor DIII-VSD. Results suggest possible FHF1A function as natural antiarrhythmic agent, that partially restore the inactivation impaired by mutations through an inactivation gate stabilization, independent of its connection to DIII-VSD.

Inhibition of hERG Potassium Channels by the Polycylic Aromatic Hydrocarbon Phenanthrene

Polycyclic aromatic hydrocarbons (PAHs) may contribute to cardiotoxic effects of hydrocarbon-based pollution. Phenanthrene (Phen) is an abundant PAH in polluted air and water. For example, Phen is a constituent PAH of crude oil spills and has been shown to increase ventricular action potential duration of fish ventricular myocytes. This action is associated with inhibition of the rapid delayed rectifier K+-current. Ether-à-go-go Related Gene encoded potassium channels (ERG) mediate IKr in multiple species including fish and human, and block of human ERG (hERG) channels by structurally diverse drugs can result in acquired long QT syndrome in humans. The purpose of this study was to investigate the action of Phen on hERG, using whole-cell patch-clamp recording from HEK 293 cells expressing wild-type (WT) or mutant hERG channels. Under conventional voltage-clamp, WT hERG current (IhERG) tails at −40 mV following an activating protocol were inhibited with an IC50 of 16.8 ± 1.7 µM, Hill slope: 1.0 ± 0.1 (mean ± SEM; n= at least 5 for 5 different concentrations). Phen (10 µM) inhibition of IhERG was voltage-dependent and was associated with leftward shift of voltage-dependent activation (V0.5 shift of −10.1 ± 0.9 mV; n=16). The extent of observed IhERG inhibition also varied with duration of the activating voltage command. IhERG carried by the attenuated inactivation N588K hERG mutant was markedly less sensitive to Phen and the F557L mutation in the S5 domain also reduced Phen inhibition. Thus, the observed IKr block with Phen appears to involve a direct inhibitory effect of the compound on (h)ERG channels that is contingent on channel gating. Intact inactivation gating and availability of the F557 residue appear to be significant influences on this inhibitory action. Funded by the British Heart Foundation PG/17/77/33125

Genetic and clinical features of SCN8A developmental and epileptic encephalopathy

ObjectiveWe aim to delineate the genetic and clinical features of SCN8A developmental and epileptic encephalopathy. MethodsNine patients with SCN8A developmental and epileptic encephalopathy were included in this study. Genetic and clinical features and effectiveness of sodium channel blockers were assessed in patients who were confirmed with SCN8A mutations. ResultsThe onset of seizures ranged from the neonatal period to 18 months of age. Seizure types were diverse and predominantly involved focal seizures or spasms. The most common initial epilepsy syndrome was West syndrome in four patients, followed by neonatal-onset focal seizures in three patients and unclassified focal epilepsy in two patients. Electroencephalograms (EEGs) showed slow and disorganized background and epileptiform abnormalities with occipital predominance. Six patients presented intractable seizures including one patient with recurrent nonconvulsive status epilepticus. Sodium channel blockers were effective in seven patients among eight patients given them. All patients showed developmental delay or regression. Severe hypotonia or ataxia was also presented in some patients. Microcephaly was also characteristic. De novo missense mutations in SCN8A were found in the inactivation gate, C-terminal, loop 2, and transmembrane segments (S1, 4, 5, and 6). There was no correlation between the location of the mutation in the protein and phenotype or response to sodium channel blockers. ConclusionSCN8A developmental and epileptic encephalopathy presents intractable seizures including spasms, focal seizures, neonatal status epilepticus, and nonconvulsive status epilepticus. Sodium channel blockers were effective irrelevant to the location of the mutation in the protein.

High-throughput phenotyping of heteromeric human ether-à-go-go-related gene potassium channel variants can discriminate pathogenic from rare benign variants

KCNH2 encodes the human ether-à-go-go-related gene potassium channel, which passes the rapid delayed rectifier potassium current. Loss-of-function variants in KCNH2 cause long QT syndrome type 2, which is associated with a markedly increased risk of cardiac arrhythmias. The majority of rare KCNH2 variants, however, are likely to be benign. The purpose of this study was to develop a high-throughput assay for discriminating pathogenic from benign KCNH2 variants. Nonsynonymous homozygous KCNH2 variants stably expressed in Flp-In human embryonic kidney 293 cell lines were phenotyped using an automated patch-clamp platform and a cell surface enzyme-linked immunosorbent assay. Functional phenotyping of heterozygous KCNH2 variants stably expressed in Flp-In human embryonic kidney 293 cell lines using a bicistronic vector was performed using an automated patch-clamp platform. In homozygous KCNH2 variant cell lines, discrepancies between current density and cell surface expression levels measured using an enzyme-linked immunosorbent assay can be explained by changes in gating properties of the variant channels. For the 30 heterozygous KCNH2 variant cell lines studied, the assay correctly predicted the ClinVar ascribed classification for 17/17 pathogenic/likely pathogenic/benign variants. Of the 13 pore-domain variants studied, 11 had a dominant-negative expression defect while the remaining 2 had enhanced inactivation gating, resulting in a dominant-negative phenotype. High-throughput electrophysiological phenotyping of heterozygous KCNH2 variants can accurately distinguish between dominant-negative, haploinsufficient loss-of-function, and benign variants. This assay will help with future classification of KCNH2 variants.

Detection of Nav1.5 Conformational Change in Mammalian Cells Using the Noncanonical Amino Acid ANAP

Nav1.5 inactivation is necessary for healthy conduction of the cardiac action potential. Genetic mutations of Nav1.5 perturb inactivation and cause potentially fatal arrhythmias associated with long QT syndrome type 3. The exact structural dynamics of the inactivation complex is unknown. To sense inactivation gate conformational change in live mammalian cells, we incorporated the solvatochromic fluorescent noncanonical amino acid 3-((6-acetylnaphthalen-2-yl)amino)-2-aminopropanoic acid (ANAP) into single sites in the Nav1.5 inactivation gate. ANAP was incorporated in full-length and C-terminally truncated Nav1.5 channels using mammalian cell synthetase-tRNA technology. ANAP-incorporated channels were expressed in mammalian cells, and they exhibited pathophysiological function. A spectral imaging potassium depolarization assay was designed to detect ANAP emission shifts associated with Nav1.5 conformational change. Site-specific intracellular ANAP incorporation affords live-cell imaging and detection of Nav1.5 inactivation gate conformational change in mammalian cells.

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

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.

A Structural Model of the Inactivation Gate of Voltage-Activated Potassium Channels

After opening, the Shaker voltage-gated potassium (KV) channel rapidly inactivates when one of its four N-termini enters and occludes the channel pore. Although it is known that the tip of the N-terminus reaches deep into the central cavity, the conformation adopted by this domain during inactivation and the nature of its interactions with the rest of the channel remain unclear. Here, we use molecular dynamics simulations coupled with electrophysiology experiments to reveal the atomic-scale mechanisms of inactivation. We find that the first six amino acids of the N-terminus spontaneously enter the central cavity in an extended conformation, establishing hydrophobic contacts with residues lining the pore. A second portion of the N-terminus, consisting of a long 24 amino acid α-helix, forms numerous polar contacts with residues in the intracellular entryway of the T1 domain. Double mutant cycle analysis revealed a strong relationship between predicted interatomic distances and empirically observed thermodynamic coupling, establishing a plausible model of the transition of KV channels to the inactivated state.

The dynamics of K+ channel gates as a biological transistor

Potassium channels are pore-forming membrane proteins that open and close in response to changes in a chemical or electrical potential, thereby regulating the flow of potassium ions across biological membranes. Two regions of the same channels are acting in tandem and enable ion flow through the channel pore. I refer to this coupled action as a “gate linker”. To closely examine the role of the gate linker in the channel function, I mutated the amino acids in the cDNA of this region, and used from knowen mutaion, either alone or together with the amino acids of adjacent regions.I have emphasized the importance of the linker between these two gates - mutations in this region may cause conformational changes that play a fundamental role in mediating the coupling between the voltage sensor, activation gate and selectivity filter elements of Kv channels. I observe that free energy considerations show the significance of the coupling between the activation and inactivation gates. Moreover, a symmetry between the coupling and sensor spring strength leads to the destruction of ion conductivity. I present a thermodynamic framework for the possible study of multiple channel blocks. The arising physical perspective of the gating process gives rise to new research avenues of the coupling mode of potassium channels and may assist in explaining the centrality of the “gate linker” to the channel function.

Activation and Inactivation Gating in Bestrophin Ion Channels

Activation and Inactivation Gating in Bestrophin Ion Channels

Screening of Negative Charges by Ca2+ in the Turret Region Controls Kv7.1 Inactivation Gating and is Regulated by PIP2 and Calmodulin

Screening of Negative Charges by Ca2+ in the Turret Region Controls Kv7.1 Inactivation Gating and is Regulated by PIP2 and Calmodulin

Using Scam to Investigate Reconfiguration of Molecular Determinants in D1-S6 during Slow Inactivation of hNav1.4

The goals of this research are to determine if 1) a single point mutation affects gating kinetics and 2) whether MTS accessibility changes via inactivation. This will be accomplished using the substituted cysteine accessibility method (SCAM) by substituting cysteine for each of the target amino acids and by applying methanethiosulfonate ethylammonium (MTSEA), which selectively binds to cysteine, indicating when the sulfhydryl side chain is accessible to binding. The target mutants consist of positions from the gating hinge to near the bundle crossing in domain 1 - segment 6 (D1-S6). At this time, data for all mutants have been recorded except for Y437C & L438C. Patch clamp techniques were used to record whole-cell currents from transiently transfected HEK cells to determine 1) the potential effects of the cysteine substitution on activation, fast inactivation, and slow inactivation, and 2) the potential accessibility of cysteine in the closed, fast inactivated, and slow inactivated states. Several mutations affected one or more baseline kinetics of the sodium channel, including significant differences in the voltage needed for either fast inactivation and/or slow inactivation in N440C, L441C, G434C, and I439C. However, MTS exposure in mutants L433C, G434C, S435C, F436C, I439C, N440C, L441C, I442C, and L443C were found to have had little-to-no effect on when the mutants were in either the closed state (−160 mV), the fast-inactivated state (brief depolarization at 0 mV), or the slow-inactivated state (prolonged depolarization at 0 mV). Thus, our results suggest that changes in accessibility of the substituted cysteines in D1-S6 of hNav1.4 may not occur, and therefore that D1-S6 may not undergo prominent reconfiguration during slow inactivation gating.

Molecular mechanisms of gating in the calcium-activated chloride channel bestrophin.

Bestrophin (BEST1-4) ligand-gated chloride (Cl-) channels are activated by calcium (Ca2+). Mutation of BEST1 causes retinal disease. Partly because bestrophin channels have no sequence or structural similarity to other ion channels, the molecular mechanisms underlying gating are unknown. Here, we present a series of cryo-electron microscopy structures of chicken BEST1, determined at 3.1 Å resolution or better, that represent the channel's principal gating states. Unlike other channels, opening of the pore is due to the repositioning of tethered pore-lining helices within a surrounding protein shell that dramatically widens a neck of the pore through a concertina of amino acid rearrangements. The neck serves as both the activation and the inactivation gate. Ca2+ binding instigates opening of the neck through allosteric means whereas inactivation peptide binding induces closing. An aperture within the otherwise wide pore controls anion permeability. The studies define a new molecular paradigm for gating among ligand-gated ion channels.