A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K(+) Channels.

Two-pore domain (K2P) K(+) channels are major regulators of excitability that endow cells with an outwardly rectifying background "leak" conductance. In some K2P channels, strong voltage-dependent activation has been observed, but the mechanism remains unresolved because they lack a canonical voltage-sensing domain. Here, we show voltage-dependent gating is common to most K2P channels and that this voltage sensitivity originates from the movement of three to four ions into the high electric field of an inactive selectivity filter. Overall, this ion-flux gating mechanism generates a one-way "check valve" within the filter because outward movement of K(+) induces filter opening, whereas inward movement promotes inactivation. Furthermore, many physiological stimuli switch off this flux gating mode to convert K2P channels into a leak conductance. These findings provide insight into the functional plasticity of a K(+)-selective filter and also refine our understanding of K2P channels and the mechanisms by which ion channels can sense voltage.

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.

KCNK18 Biallelic Variants Associated with Intellectual Disability and Neurodevelopmental Disorders Alter TRESK Channel Activity.

The TWIK-related spinal cord potassium channel (TRESK) is encoded by KCNK18, and variants in this gene have previously been associated with susceptibility to familial migraine with aura (MIM #613656). A single amino acid substitution in the same protein, p.Trp101Arg, has also been associated with intellectual disability (ID), opening the possibility that variants in this gene might be involved in different disorders. Here, we report the identification of KCNK18 biallelic missense variants (p.Tyr163Asp and p.Ser252Leu) in a family characterized by three siblings affected by mild-to-moderate ID, autism spectrum disorder (ASD) and other neurodevelopment-related features. Functional characterization of the variants alone or in combination showed impaired channel activity. Interestingly, Ser252 is an important regulatory site of TRESK, suggesting that alteration of this residue could lead to additive downstream effects. The functional relevance of these mutations and the observed co-segregation in all the affected members of the family expand the clinical variability associated with altered TRESK function and provide further insight into the relationship between altered function of this ion channel and human disease.

Altered functional properties of a missense variant in the TRESK K+ channel (KCNK18) associated with migraine and intellectual disability.

Mutations in the KCNK18 gene that encodes the TRESK K2P potassium channel have previously been linked with typical familial migraine with aura. Recently, an atypical clinical case has been reported in which a male individual carrying the p.Trp101Arg (W101R) missense mutation in the KCNK18 gene was diagnosed with intellectual disability and migraine with brainstem aura. Here we report the functional characterization of this new missense variant. This mutation is located in a highly conserved residue close to the selectivity filter, and our results show although these mutant channels retain their K+ selectivity and calcineurin-dependent regulation, the variant causes an overall dramatic loss of TRESK channel function as well as an initial dominant-negative effect when co-expressed with wild-type channels in Xenopus laevis oocytes. The dramatic functional consequences of this mutation thereby support a potentially pathogenic role for this variant and provide further insight into the relationship between the structure and function of this ion channel.

Association of A Novel Splice Site Mutation in P/Q-Type Calcium Channels with Childhood Epilepsy and Late-Onset Slowly Progressive Non-Episodic Cerebellar Ataxia.

Episodic ataxia type 2 (EA2) is characterized by paroxysmal attacks of ataxia with typical onset in childhood or early adolescence. The disease is associated with mutations in the voltage-gated calcium channel alpha 1A subunit (Cav2.1) that is encoded by the CACNA1A gene. However, previously unrecognized atypical symptoms and the genetic overlap existing between EA2, spinocerebellar ataxia type 6, familial hemiplegic migraine type 1, and other neurological diseases blur the genotype/phenotype correlations, making a differential diagnosis difficult to formulate correctly and delaying early therapeutic intervention. Here we report a new clinical phenotype of a CACNA1A-associated disease characterized by absence epilepsy occurring during childhood. However, much later in life the patient displayed non-episodic, slowly progressive gait ataxia. Gene panel sequencing for hereditary ataxias led to the identification of a novel heterozygous CACNA1A mutation (c.1913 + 2T > G), altering the donor splice site of intron 14. This genetic defect was predicted to result in an in-frame deletion removing 44 amino acids from the voltage-gated calcium channel Cav2.1. An RT-PCR analysis of cDNA derived from patient skin fibroblasts confirmed the skipping of the entire exon 14. Furthermore, two-electrode voltage-clamp recordings performed from Xenopus laevis oocytes expressing a wild-type versus mutant channel showed that the genetic defect caused a complete loss of channel function. This represents the first description of distinct clinical manifestations that remarkably expand the genetic and phenotypic spectrum of CACNA1A-related diseases and should be considered for an early diagnosis and effective therapeutic intervention.

Sodium channels as gateable non-photonic sensors for membrane-delimited reactive species.

Reactive oxygen species (ROS) and reactive oxygen intermediates (ROI) play crucial roles in physiological processes. While excessive ROS damages cells, small fluctuations in ROS levels represent physiological signals important for vital functions. Despite the physiological importance of ROS, many fundamental questions remain unanswered, such as which types of ROS occur in cells, how they distribute inside cells, and how long they remain in an active form. The current study presents a ratiometric sensor of intracellular ROS levels based on genetically engineered voltage-gated sodium channels (roNaV). roNaV can be used for detecting oxidative modification that occurs near the plasma membrane with a sensitivity similar to existing fluorescence-based ROS sensors. Moreover, roNaV has several advantages over traditional sensors because it does not need excitation light for sensing, and thus, can be used to detect phototoxic cellular modifications. In addition, the ROS dynamic range of roNaV is easily manipulated in real time by means of the endogenous channel inactivation mechanism. Measurements on ROS liberated from intracellular Lucifer Yellow and genetically encoded KillerRed have revealed an assessment of ROS lifetime in individual mammalian cells. Flashlight-induced ROS concentration decayed with two major time constants of about 10 and 1000 ms.

Heterologous expression of NaV1.9 chimeras in various cell systems.

SCN11A encodes the voltage-gated sodium channel NaV1.9, which deviates most strongly from the other eight NaV channels expressed in mammals. It is characterized by resistance to the prototypic NaV channel blocker tetrodotoxin and exhibits slow activation and inactivation gating. Its expression in dorsal root ganglia neurons suggests a role in motor or pain signaling functions as also recently demonstrated by the occurrence of various mutations in human SCN11A leading to altered pain sensation syndromes. The systematic investigation of human NaV1.9, however, is severely hampered because of very poor heterologous expression in host cells. Using patch-clamp and two-electrode voltage-clamp methods, we show that this limitation is caused by the C-terminal structure of NaV1.9. A chimera of NaV1.9 harboring the C terminus of NaV1.4 yields functional expression not only in neuronal cells but also in non-excitable cells, such as HEK 293T or Xenopus oocytes. The major functional difference of the chimeric channel with respect to NaV1.9 is an accelerated activation and inactivation. Since the entire transmembrane domain is preserved, it is suited for studying pharmacological properties of the channel and the functional impact of disease-causing mutations. Moreover, we demonstrate how mutation S360Y makes NaV1.9 channels sensitive to tetrodotoxin and saxitoxin and that the unusual slow open-state inactivation of NaV1.9 is also mediated by the IFM (isoleucine-phenylalanine-methionine) inactivation motif located in the linker connecting domains III and IV.

Side pockets provide the basis for a new mechanism of Kv channel-specific inhibition.

Most known small-molecule inhibitors of voltage-gated ion channels have poor subtype specificity because they interact with a highly conserved binding site in the central cavity. Using alanine-scanning mutagenesis, electrophysiological recordings and molecular modeling, we have identified a new drug-binding site in Kv1.x channels. We report that Psora-4 can discriminate between related Kv channel subtypes because, in addition to binding the central pore cavity, it binds a second, less conserved site located in side pockets formed by the backsides of S5 and S6, the S4-S5 linker, part of the voltage sensor and the pore helix. Simultaneous drug occupation of both binding sites results in an extremely stable nonconducting state that confers high affinity, cooperativity, use-dependence and selectivity to Psora-4 inhibition of Kv1.x channels. This new mechanism of inhibition represents a molecular basis for the development of a new class of allosteric and selective voltage-gated channel inhibitors.

The effects of stretch activation on ionic selectivity of the TREK-2 K2P K+ channel.

The TREK-2 (KCNK10) K2P potassium channel can be regulated by variety of polymodal stimuli including pressure. In a recent study, we demonstrated that this mechanosensitive K+ channel responds to changes in membrane tension by undergoing a major structural change from its 'down' state to the more expanded 'up' state conformation. These changes are mostly restricted to the lower part of the protein within the bilayer, but are allosterically coupled to the primary gating mechanism located within the selectivity filter. However, any such structural changes within the filter also have the potential to alter ionic selectivity and there are reports that some K2Ps, including TREK channels, exhibit a dynamic ionic selectivity. In this addendum to our previous study we have therefore examined whether the selectivity of TREK-2 is altered by stretch activation. Our results reveal that the filter remains stable and highly selective for K+ over Na+ during stretch activation, and that permeability to a range of other cations (Rb+, Cs+ and NH4+) also does not change. The asymmetric structural changes that occur during stretch activation therefore allow the channel to respond to changes in membrane tension without a loss of K+ selectivity.

Insights into the structural nature of the transition state in the Kir channel gating pathway.

In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a 'catalytic' effect upon the channel gating kinetics.