Use-dependent potentiation of the Nav1.6 sodium channel

Nav1.2 and Nav1.6 are two voltage-gated sodium channel isoforms that are abundant in the adult central nervous system. These channels are expressed in different cells and localized in different neuronal regions, which may reflect functional specialization. To examine this possibility, we compared the properties of Nav1.2 and Nav1.6 in response to a rapid series of repetitive depolarizations. Currents through Nav1.6 coexpressed with beta1 demonstrated use-dependent potentiation during a rapid train of depolarizations. This potentiation was in contrast to the use-dependent decrease in current for Nav1.2 with beta1. The voltage dependence of potentiation correlated with the voltage dependence of activation, and it still occurred when fast inactivation was removed by mutation. Rapid stimulation accelerated a slow phase of activation in the Nav1.6 channel that had fast inactivation removed, resulting in faster channel activation. Although the Nav1.2 channel with fast inactivation removed also demonstrated slightly faster activation, that channel showed very pronounced slow inactivation compared to Nav1.6. These results indicate that potentiation of Nav1.6 sodium currents results from faster channel activation, and that this effect is masked by slow inactivation in Nav1.2. The data suggest that Nav1.6 might be more resistant to inactivation, which might be helpful for high-frequency firing at nodes of Ranvier compared to Nav1.2.     

Positive Allosteric Modulation of Kv Channels by Sevoflurane: Insights into the Structural Basis of Inhaled Anesthetic Action

Inhalational general anesthesia results from the poorly understood interactions of haloethers with multiple protein targets, which prominently includes ion channels in the nervous system. Previously, we reported that the commonly used inhaled anesthetic sevoflurane potentiates the activity of voltage-gated K⁺ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K-Shaw2 channel. Also, previous work suggested that the S4-S5 linker of K-Shaw2 plays a role in the inhibition of this Kv channel by n-alcohols and inhaled anesthetics. Here, we hypothesized that the S4-S5 linker is also a determinant of the potentiation of Kv1.2 and K-Shaw2 by sevoflurane. Following functional expression of these Kv channels in Xenopus oocytes, we found that converse mutations in Kv1.2 (G329T) and K-Shaw2 (T330G) dramatically enhance and inhibit the potentiation of the corresponding conductances by sevoflurane, respectively. Additionally, Kv1.2-G329T impairs voltage-dependent gating, which suggests that Kv1.2 modulation by sevoflurane is tied to gating in a state-dependent manner. Toward creating a minimal Kv1.2 structural model displaying the putative sevoflurane binding sites, we also found that the positive modulations of Kv1.2 and Kv1.2-G329T by sevoflurane and other general anesthetics are T1-independent. In contrast, the positive sevoflurane modulation of K-Shaw2 is T1-dependent. In silico docking and molecular dynamics-based free-energy calculations suggest that sevoflurane occupies distinct sites near the S4-S5 linker, the pore domain and around the external selectivity filter. We conclude that the positive allosteric modulation of the Kv channels by sevoflurane involves separable processes and multiple sites within regions intimately involved in channel gating.     

Brownian dynamics simulations of the recognition of the scorpion toxin maurotoxin with the voltage-gated potassium ion channels

The recognition of the scorpion toxin maurotoxin (MTX) by the voltage-gated potassium (Kv1) channels, Kv1.1, Kv1.2, and Kv1.3, has been studied by means of Brownian dynamics (BD) simulations. All of the 35 available structures of MTX in the Protein Data Bank (http://www.rcsb.org/pdb) determined by nuclear magnetic resonance were considered during the simulations, which indicated that the conformation of MTX significantly affected both the recognition and the binding between MTX and the Kv1 channels. Comparing the top five highest-frequency structures of MTX binding to the Kv1 channels, we found that the Kv1.2 channel, with the highest docking frequencies and the lowest electrostatic interaction energies, was the most favorable for MTX binding, whereas Kv1.1 was intermediate, and Kv1.3 was the least favorable one. Among the 35 structures of MTX, the 10th structure docked into the binding site of the Kv1.2 channel with the highest probability and the most favorable electrostatic interactions. From the MTX-Kv1.2 binding model, we identified the critical residues for the recognition of these two proteins through triplet contact analyses. MTX locates around the extracellular mouth of the Kv1 channels, making contacts with its beta-sheets. Lys23, a conserved amino acid in the scorpion toxins, protrudes into the pore of the Kv1.2 channel and forms two hydrogen bonds with the conserved residues Gly401(D) and Tyr400(C) and one hydrophobic contact with Gly401(C) of the Kv1.2 channel. The critical triplet contacts for recognition between MTX and the Kv1.2 channel are Lys23(MTX)-Asp402(C)(Kv1), Lys27(MTX)-Asp378(D)(Kv1), and Lys30(MTX)-Asp402(A)(Kv1). In addition, six hydrogen-bonding interactions are formed between residues Lys23, Lys27, Lys30, and Tyr32 of MTX and residues Gly401, Tyr400, Asp402, Asp378, and Thr406 of Kv1.2. Many of them are formed by side chains of residues of MTX and backbone atoms of the Kv1.2 channel. Five hydrophobic contacts exist between residues Pro20, Lys23, Lys30 and Tyr32 of MTX and residues Asp402, Val404, Gly401, and Arg377 of the Kv1.2 channel. The simulation results are in agreement with the previous molecular biology experiments and explain the binding phenomena between MTX and Kv1 channels at the molecular level. The consistency between the results of the BD simulations and the experimental data indicated that our three-dimensional model of the MTX-Kv1.2 channel complex is reasonable and can be used in additional biological studies, such as rational design of novel therapeutic agents blocking the voltage-gated channels and in mutagenesis studies in both the toxins and the Kv1 channels. In particular, both the BD simulations and the molecular mechanics refinements indicate that residue Asp378 of the Kv1.2 channel is critical for its recognition and binding functionality toward MTX. This phenomenon has not been appreciated in the previous mutagenesis experiments, indicating this might be a new clue for additional functional study of Kv1 channels.     

Kv beta subunit oxidoreductase activity and Kv1 potassium channel trafficking.

Voltage-gated Kv1 potassium channels consist of pore-forming alpha subunits and cytoplasmic Kv beta subunits. The latter play diverse roles in modulating the gating, stability, and trafficking of Kv1 channels. The crystallographic structure of the Kv beta2 subunit revealed surprising structural homology with aldo-keto reductases, including a triosephosphate isomerase barrel structure, conservation of key catalytic residues, and a bound NADP(+) cofactor (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell 90, 943-952). Each Kv1-associated Kv beta subunit (Kv beta 1.1, Kv beta 1.2, Kv beta 2, and Kv beta 3) shares striking amino acid conservation in key catalytic and cofactor binding residues. Here, by a combination of structural modeling and biochemical and cell biological analyses of structure-based mutations, we investigate the potential role for putative Kv beta subunit enzymatic activity in the trafficking of Kv1 channels. We found that all Kv beta subunits promote cell surface expression of coexpressed Kv1.2 alpha subunits in transfected COS-1 cells. Kv beta1.1 and Kv beta 2 point mutants lacking a key catalytic tyrosine residue found in the active site of all aldo-keto reductases have wild-type trafficking characteristics. However, mutations in residues within the NADP(+) binding pocket eliminated effects on Kv1.2 trafficking. In cultured hippocampal neurons, Kv beta subunit coexpression led to axonal targeting of Kv1.2, recapitulating the Kv1.2 localization observed in many brain neurons. Similar to the trafficking results in COS-1 cells, mutations within the cofactor binding pocket reduced axonal targeting of Kv1.2, whereas those in the catalytic tyrosine did not. Together, these data suggest that NADP(+) binding and/or the integrity of the binding pocket structure, but not catalytic activity, of Kv beta subunits is required for intracellular trafficking of Kv1 channel complexes in mammalian cells and for axonal targeting in neurons.     

Characterization of a new Kv1.3 channel-specific blocker, J123, from the scorpion Buthus martensii Karsch

The potassium channel Kv1.3 is an attractive pharmacological target for T-cell-mediated autoimmune diseases, and specific and selective peptidic blockers of Kv1.3 channels have served as valuable therapeutic leads for treating these diseases. Here, we found a new peptide toxin, J123, with 43 amino acids including six cysteine residues by screening the venomous cDNA library of scorpion Buthus martensii Karsch, which has been used as traditional medicine in China for more than 2000 years. The sequence analysis suggested that peptide J123 constituted a new member of the alpha-KTx toxins. The electrophysiological experiments further indicated that peptide J123 has a novel pharmacological profiles: it blocked Kv1.3 channel with high potency (IC(50)=0.79nM), and exhibited good selectivity on Kv1.3 over Kv1.1 (>1000-fold) and Kv1.2 (about 30-fold), respectively. Furthermore, peptide J123 had no activity on SKCa2 and SKCa3 channels at micromolar concentration level. Based on the pharmacological activities, the possible channel-interacting surface of peptide J123 was also predicted by molecular modeling and docking. All these data not only enrich the knowledge of the structure-function relationship of the new Kv1.3-speicific peptide but also present a potential drug candidate for selectively targeting Kv1.3 channels.     

Regulatory role of the extreme C‐terminal end of the S6 inner helix in C‐terminal‐truncated Kv1.2 channel activation

The transmembrane part of the S6 inner helix of the Kv1.2 potassium channel is a pivotal part in sustaining channel activity. However, the role of its extreme C‐terminal end, which is located on the cytoplasmic side of the channel, is largely unknown. Here, we investigated the role of the extreme C‐terminal end of the S6 inner helix (the HRET region) by constructing a series of C‐terminal‐truncated mutations related to this region in the C‐terminal‐truncated Kv1.2 channel. Mutations on Thr⁴²¹ or Glu⁴²⁰ significantly altered the activation of the truncated channel. Mutations on Arg⁴¹⁹ demonstrated that neutral or basic, but not acidic amino acid, is essential at the position for the truncated channel activation, and no functional channel was observed when the channel was truncated from His⁴¹⁸. Hence, our results indicate that the extreme C‐terminal end of the S6 inner helix plays an important regulatory role in the activation of the C‐terminal‐truncated Kv1.2 channel.     

Differential modulation of Kv1 channel-mediated currents by co-expression of Kvbeta3 subunit in a mammalian cell-line

The effect of Kvbeta3 subunit co-expression on currents mediated by the Shaker-related channels Kv1.1 to Kv1.6 in Chinese hamster ovary (CHO) cells was studied with patch-clamp techniques. In the presence of Kvbeta3, differences in the voltage dependence of activation for Kv1.1, Kv1.3 and Kv1.6 were detected, but not for Kv1.2- and Kv1.4-mediated currents. Co-expression of Kvbeta3 did not cause a significant increase in current density for any of the tested channels. In contrast to previous studies in Xenopus oocyte expression system, Kvbeta3 confered a rapid inactivation to all except Kv1.3 channels. Also, Kv1.6 channels that possess an N-type inactivation prevention (NIP) domain for Kvbeta1.1, inactivated rapidly when co-expressed with Kvbeta3. Onset and recovery kinetics of channel inactivation distinctly differed for the various Kv1alpha/Kvbeta3 subunit combinations investigated in this study. The results indicate that the choice of expression system may critically determine Kvbeta3 inactivating activity. This suggests that the presence of an inactivating domain and a receptor in a channel pore, although necessary, may not be sufficient for an effective rapid N-type inactivation of Kv1 channels in heterologous expression systems.     

Mutations in the KCNA1 gene associated with episodic ataxia type-1 syndrome impair heteromeric voltage-gated K(+) channel function.

Episodic ataxia type-1 syndrome (EA-1) is an autosomal dominant neurological disorder that manifests itself during infancy and results from point mutations in the voltage-gated potassium channel gene hKv1.1. The hallmark of the disease is continuous myokymia and episodic attacks of spastic contractions of the skeletal muscles, which cause permanent disability. Coexpression of hKv1.1 and hKv1.2 subunits produces heteromeric potassium channels with biophysical and pharmacological properties intermediate between the respective homomers. By using tandemly linked subunits, we demonstrate that hKv1.1 subunits bearing the EA-1 mutations V408A and E325D combine with hKv1.2 to produce channels with altered kinetics of activation, deactivation, C-type inactivation, and voltage dependence. Moreover, hKv1.1V408A single-channel analysis reveals a approximately threefold reduction of the mean open duration of the channel compared with the wild-type, and this mutation alters the open-state stability of both homomeric and heteromeric channels. The results demonstrate that human Kv1.2 and Kv1.1 subunits coassemble to form a novel channel with distinct gating properties that are altered profoundly by EA-1 mutations, thus uncovering novel physiopathogenetic mechanisms of episodic ataxia type-1 myokymia syndrome.     

Length-dependent regulation of the Kv1.2 channel activation by its C-terminus

The cytoplasmic C-terminus plays regulatory roles in the gating of many ion channels. However, lack of structural information on the C-terminus prevents the elucidation of how the C-terminal domain interacts with the gating machinery to exert its effects on the channel gating. In this report, we investigated the regulatory role of the C-terminus with functional study and structural modeling of a succession of C-terminal truncations of the Kv1.2 and Kv1.2₄₂₇-KcsA_112-160 chimeric channels. Functional study demonstrated a length-dependent shift of the activation curves for the C-terminal truncations of the Kv1.2 channel. Structural modeling indicated that the C-terminus of one subunit could dynamically interact with the S4–S5 linker of a neighboring subunit and the probability of interaction was dependent on the length of the C-terminal truncated Kv1.2 channels. In contrast, no length-dependent shift of the activation curve and probability of interaction between C-terminus and the neighboring S4–S5 linker were observed for the truncations of the Kv1.2-KcsA chimeric channel, suggesting that the native C-terminus of the Kv1.2 channel is essential for the interaction. Furthermore, surface plasmon resonance measurements indicated that there is direct interaction between the C-terminal domain and the S4–S5 linker of the Kv1.2 channel. These results imply that the dynamic interaction of the C-terminus with the S4–S5 linker from a neighboring subunit of the Kv1.2 channel provides a mechanism for its C-terminus to regulate the channel activation.     

Multifaceted Modulation of K+ Channels by Protein-tyrosine Phosphatase {epsilon} Tunes Neuronal Excitability

Non-receptor-tyrosine kinases (protein-tyrosine kinases) and non-receptor tyrosine phosphatases (PTPs) have been implicated in the regulation of ion channels, neuronal excitability, and synaptic plasticity. We previously showed that protein-tyrosine kinases such as Src kinase and PTPs such as PTPα and PTPε modulate the activity of delayed-rectifier K(+) channels (I(K)). Here we show cultured cortical neurons from PTPε knock-out (EKO) mice to exhibit increased excitability when compared with wild type (WT) mice, with larger spike discharge frequency, enhanced fast after-hyperpolarization, increased after-depolarization, and reduced spike width. A decrease in I(K) and a rise in large-conductance Ca(2+)-activated K(+) currents (mBK) were observed in EKO cortical neurons compared with WT. Parallel studies in transfected CHO cells indicate that Kv1.1, Kv1.2, Kv7.2/7.3, and mBK are plausible molecular correlates of this multifaceted modulation of K(+) channels by PTPε. In CHO cells, Kv1.1, Kv1.2, and Kv7.2/7.3 K(+) currents were up-regulated by PTPε, whereas mBK channel activity was reduced. The levels of tyrosine phosphorylation of Kv1.1, Kv1.2, Kv7.3, and mBK potassium channels were increased in the brain cortices of neonatal and adult EKO mice compared with WT, suggesting that PTPε in the brain modulates these channel proteins. Our data indicate that in EKO mice, the lack of PTPε-mediated dephosphorylation of Kv1.1, Kv1.2, and Kv7.3 leads to decreased I(K) density and enhanced after-depolarization. In addition, the deficient PTPε-mediated dephosphorylation of mBK channels likely contributes to enhanced mBK and fast after-hyperpolarization, spike shortening, and consequent increase in neuronal excitability observed in cortical neurons from EKO mice.     

Molecular restraints in the permeation pathway of ion channels

Ion channels assist and control the diffusion of ions through biological membranes. The conduction process depends on the structural characteristics of these nanopores, among which are the hydrophobicity and the afforded diameter of the conduction pathway. In this contribution, we use full atomistic free-energy molecular dynamics simulations to estimate the effect of such characteristics on the energetics of ion conduction through the activation gate of voltage-gated potassium (Kv) channels. We consider specifically the ionic translocation through three different permeation pathways, corresponding to the activation gate of an atomistic model of Shaker channels in closed and partially opened conformations, and that of the open conformation of the Kv1.2 channel. In agreement with experiments, we find that the region of Val(478) constitutes the main gate. The conduction is unfavorable through this gate when the constriction is smaller than an estimated threshold of 4.5-5.0 A, mainly due to incomplete coordination-hydration of the ion. Above this critical size, e.g., for the Kv1.2, the valine gate is wide enough to allow fully coordination of the ion and therefore its diffusion on a flat energy surface. Similar to other ion channels, Kv channels appear therefore to regulate diffusion by constricting hydrophobic regions of the permeation pathway.     

Effect of sensor domain mutations on the properties of voltage-gated ion channels: molecular dynamics studies of the potassium channel Kv1.2

The effects on the structural and functional properties of the Kv1.2 voltage-gated ion channel, caused by selective mutation of voltage sensor domain residues, have been investigated using classical molecular dynamics simulations. Following experiments that have identified mutations of voltage-gated ion channels involved in state-dependent omega currents, we observe for both the open and closed conformations of the Kv1.2 that specific mutations of S4 gating-charge residues destabilize the electrostatic network between helices of the voltage sensor domain, resulting in the formation of hydrophilic pathways linking the intra- and extracellular media. When such mutant channels are subject to transmembrane potentials, they conduct cations via these so-called “omega pores.” This study provides therefore further insight into the molecular mechanisms that lead to omega currents, which have been linked to certain channelopathies.     

Molecular cloning and functional characterization of a novel delayed rectifier potassium channel from channel catfish (Ictalurus punctatus): expression in taste buds

The gustatory system of channel catfish is widely studied for its sensitivity to amino acids. As a first step in identifying the molecular components that play a role in taste transduction in catfish, we cloned the full-length cDNA for Kv2-catfish, a novel K(+) channel that is expressed in taste buds. The deduced amino acid sequence is 816 residues, and shares a 56-59% sequence identity with Kv2.1 and Kv2.2, the other members of the vertebrate Kv2 subfamily of voltage-gated K(+) channels. The Kv2-catfish RNA was expressed in taste buds, brain, skeletal muscle, kidney, intestine and gills, and its gene is represented as a single copy in the catfish genome. Recombinant channels expressed in XENOPUS: oocytes were selective for K(+), and were inhibited by tetraethylammonium applied to the extracellular side of the membrane during two-electrode voltage clamp analysis with a 50% inhibitory constant of 6.1 mM. The channels showed voltage-dependent activation, and did not inactivate within 200 ms. Functionally, Kv2-catfish is a voltage-gated, delayed rectifier K(+) channel, and its primary structure is the most divergent sequence identified among the vertebrate members of the Kv2 subfamily of K(+) channels, being related equally well to Kv2.1 and Kv2.2.     

Molecular basis of dysfunctional Kv channels in small coronary artery smooth muscle cells of streptozotocin-induced diabetic rats

We have previously shown that diabetes impaired cAMP-mediated endothelium independent vasodilation of rat small coronary arteries. Inhibition of Kv channel activity plays an important role in the decrease of cAMP mediated vasodilation. The present study investigated the effect of streptozotocin (STZ)-induced diabetes on mRNA and protein expressions of Kv1.2 and Kv1.5 channels in vascular smooth muscle cells of rat small coronary artery using RT-PCR, Western blot and immunohistochemistry methods. STZ-induced diabetes obviously impaired mRNA expression of Kv1.2 and Kv1.5 channel. The mRNA levels of Kv1.2 channel were 0.65 +/- 0.08 and 1.02 +/- 0.17 in STZ rats and control rats, respectively (n = 7, P < 0.05). Whereas the levels of Kv1.5 channel were 0.58 +/- 0.05 and 0.94 +/- 0.13 in STZ rats and control rats, respectively (n = 7, P < 0.05). Western blotting analysis showed that protein expression of Kv1.2 channel was decreased significantly but not Kv1.5 channel. Protein expressions of Kv1.2 channel were 0.49 +/- 0.04 and 0.70 +/- 0.06 in STZ rats and control rats, respectively (n = 5, P < 0.05), but those of Kv1.5 channel were 0.61 +/- 0.12 and 0.59 +/- 0.14 in STZ rats and control rats, respectively (n = 5, P > 0.05). Immunohistochemistry identification indicated that immunological reaction of Kv1.2 channel protein was attenuated, but Kv1.5 channel protein was not altered. Positive staining intensity normalized by gray values of Kv1.2 channel were 173 +/- 13 and 131 +/- 11 in STZ rats and control rats, respectively (n = 5, P < 0.05), but those of Kv1.5 channel were 139 +/- 16 and 141 +/- 12 in STZ rats and control rats, respectively (n = 5, P > 0.05). These results suggested that impairment of cAMP-mediated endothelium independent vasodilation of rat small coronary artery by STZ-induced diabetes was resulted from decrease of mRNA and protein expressions of Kv channels, and which eventually leads to a reduced current from Kv channels.     

Use-dependent block of the voltage-gated Na+ channel by tetrodotoxin and saxitoxin: Effect of pore mutations that change ionic selectivity

Voltage-gated Na⁺ channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca²⁺ permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca²⁺ or Na⁺ ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca²⁺ permeability, suggesting that ion–toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.     

Functional Analysis of Missense Mutations in Kv8.2 Causing Cone Dystrophy with Supernormal Rod Electroretinogram

Mutations in KCNV2 have been proposed as the molecular basis for cone dystrophy with supernormal rod electroretinogram. KCNV2 codes for the modulatory voltage-gated potassium channel α-subunit, Kv8.2, which is incapable of forming functional channels on its own. Functional heteromeric channels are however formed with Kv2.1 in heterologous expression systems, with both α-subunit genes expressed in rod and cone photoreceptors. Of the 30 mutations identified in the KCNV2 gene, we have selected three missense mutations localized in the potassium channel pore and two missense mutations localized in the tetramerization domain for analysis. We characterized the differences between homomeric Kv2.1 and heteromeric Kv2.1/Kv8.2 channels and investigated the influence of the selected mutations on the function of heteromeric channels. We found that two pore mutations (W467G and G478R) led to the formation of nonconducting heteromeric Kv2.1/Kv8.2 channels, whereas the mutations localized in the tetramerization domain prevented heteromer generation and resulted in the formation of homomeric Kv2.1 channels only. Consequently, our study suggests the existence of two distinct molecular mechanisms involved in the disease pathology.     

A voltage-dependent gating transition induces use-dependent block by tetrodotoxin of rat IIA sodium channels expressed in Xenopus oocytes.

We have utilized molecular biological techniques to demonstrate that rat IIA sodium channels expressed in Xenopus oocytes were blocked by tetrodotoxin (TTX) in a use-dependent manner. This use dependence was the result of an increased affinity of the channels for TTX upon depolarization, most likely due to a conformational change in the channel. Using a mutant with a slower macroscopic rate of inactivation, we have demonstrated that this conformational change is not the transition into the fast-inactivated state. The transition is probably one occurring during activation of the channel, as suggested by the fact that one sodium channel mutant demonstrated comparable depolarizing shifts in the voltage dependence of both activation and use-dependent block by TTX. The transition occurred at potentials more negative than those resulting in channel conductance, suggesting that the conformational change that causes use-dependent block by TTX is a closed-state voltage-dependent gating transition.