Molecular and Functional Differences between Heart mKv1.7 Channel Isoforms

Ion channels are membrane-spanning proteins that allow ions to permeate at high rates. The kinetic characteristics of the channels present in a cell determine the cell signaling profile and therefore cell function in many different physiological processes. We found that Kv1.7 channels from mouse heart muscle have two putative translation initiation start sites that generate two channel isoforms with different functional characteristics, mKv1.7L (489 aa) and a shorter mKv1.7S (457 aa). The electrophysiological analysis of mKv1.7L and mKv1.7S channels revealed that the two channel isoforms have different inactivation kinetics. The channel resulting from the longer protein (L) inactivates faster than the shorter channels (S). Our data supports the hypothesis that mKv1.7L channels inactivate predominantly due to an N-type related mechanism, which is impaired in the mKv1.7S form. Furthermore, only the longer version mKv1.7L is regulated by the cell redox state, whereas the shorter form mKv1.7S is not. Thus, expression starting at each translation initiation site results in significant functional divergence. Our data suggest that the redox modulation of mKv1.7L may occur through a site in the cytoplasmic N-terminal domain that seems to encompass a metal coordination motif resembling those found in many redox-sensitive proteins. The mRNA expression profile and redox modulation of mKv1.7 kinetics identify these channels as molecular entities of potential importance in cellular redox-stress states such as hypoxia

Marine Toxins Targeting Kv1 Channels: Pharmacological Tools and Therapeutic Scaffolds

Toxins from marine animals provide molecular tools for the study of many ion channels, including mammalian voltage-gated potassium channels of the Kv1 family. Selectivity profiling and molecular investigation of these toxins have contributed to the development of novel drug leads with therapeutic potential for the treatment of ion channel-related diseases or channelopathies. Here, we review specific peptide and small-molecule marine toxins modulating Kv1 channels and thus cover recent findings of bioactives found in the venoms of marine Gastropod (cone snails), Cnidarian (sea anemones), and small compounds from cyanobacteria. Furthermore, we discuss pivotal advancements at exploiting the interaction of κM-conotoxin RIIIJ and heteromeric Kv1.1/1.2 channels as prevalent neuronal Kv complex. RIIIJ’s exquisite Kv1 subtype selectivity underpins a novel and facile functional classification of large-diameter dorsal root ganglion neurons. The vast potential of marine toxins warrants further collaborative efforts and high-throughput approaches aimed at the discovery and profiling of Kv1-targeted bioactives, which will greatly accelerate the development of a thorough molecular toolbox and much-needed therapeutics

NMR structure of μ-conotoxin GIIIC : leucine 18 induces local repacking of the N-terminus resulting in reduced NaV channel potency

mu-Conotoxins are potent and highly specific peptide blockers of voltage-gated sodium channels. In this study, the solution structure of mu-conotoxin GIIIC was determined using 2D NMR spectroscopy and simulated annealing calculations. Despite high sequence similarity, GIIIC adopts a three-dimensional structure that differs from the previously observed conformation of mu-conotoxins GIIIA and GIIIB due to the presence of a bulky, non-polar leucine residue at position 18. The side chain of L18 is oriented towards the core of the molecule and consequently the N-terminus is re-modeled and located closer to L18. The functional characterization of GIIIC defines it as a canonical mu-conotoxin that displays substantial selectivity towards skeletal muscle sodium channels (Na-V), albeit with similar to 2.5-fold lower potency than GIIIA. GIIIC exhibited a lower potency of inhibition of Na(V)1.4 channels, but the same Na-V selectivity profile when compared to GIIIA. These observations suggest that single amino acid differences that significantly affect the structure of the peptide do in fact alter its functional properties. Our work highlights the importance of structural factors, beyond the disulfide pattern and electrostatic interactions, in the understanding of the functional properties of bioactive peptides. The latter thus needs to be considered when designing analogues for further applications

Changes in single K+ channel behavior through the lipid phase transition

We show that the activity of an ion channel is strictly related to the phase state of the lipid bilayer hosting the channel. By measuring unitary conductance, dwell times, and open probability of the K+ channel KcsA as a function of temperature in lipid bilayers composed of POPE and POPG in different relative proportions, we obtain that all those properties show a trend inversion when the bilayer is in the transition region between the liquid disordered and the solid ordered phase. These data suggest that the physical properties of the lipid bilayer influence ion channel activity likely via a fine tuning of its conformations. In a more general interpretative framework, we suggest that other parameters such as pH, ionic strength, and the action of amphiphilic drugs can affect the physical behavior of the lipid bilayer in a fashion similar to temperature changes resulting in functional changes of transmembrane proteins

Pharmacological and genetic characterisation of the canine P2X4 receptor

Background and Purpose: P2X4 receptors are emerging therapeutic targets for treating chronic pain and cardiovascular disease. Dogs are well-recognised natural models of human disease, but information regarding P2X4 receptors in dogs is lacking. To aid the development and validation of P2X4 receptor ligands, we have characterised and compared canine and human P2X4 receptors. Experimental Approach: Genomic DNA was extracted from whole blood samples from 101 randomly selected dogs and sequenced across the P2RX4 gene to identify potential missense variants. Recombinant canine and human P2X4 receptors tagged with Emerald GFP were expressed in 1321N1 and HEK293 cells and analysed by immunoblotting and confocal microscopy. In these cells, receptor pharmacology was characterised using nucleotide-induced Fura-2 AM measurements of intracellular Ca 2+ and known P2X4 receptor antagonists. P2X4 receptor-mediated inward currents in HEK293 cells were assessed by automated patch clamp. Key Results: No P2RX4 missense variants were identified in any canine samples. Canine and human P2X4 receptors were localised primarily to lysosomal compartments. ATP was the primary agonist of canine P2X4 receptors with near identical efficacy and potency at human receptors. 2′(3′)-O-(4-benzoylbenzoyl)-ATP, but not ADP, was a partial agonist with reduced potency for canine P2X4 receptors compared to the human orthologues. Five antagonists inhibited canine P2X4 receptors, with 1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea displaying reduced sensitivity and potency at canine P2X4 receptors. Conclusion and Implications: P2X4 receptors are highly conserved across dog pedigrees and display expression patterns and pharmacological profiles similar to human receptors, supporting validation and use of therapeutic agents for P2X4 receptor-related disease onset and management in dogs and humans

Structural model of the open-closed-inactivated cycle of prokaryotic voltage-gated sodium channels

In excitable cells, the initiation of the action potential results from the opening of voltage-gated sodium channels. These channels undergo a series of conformational changes between open, closed, and inactivated states. Many models have been proposed for the structural transitions that result in these different functional states. Here, we compare the crystal structures of prokaryotic sodium channels captured in the different conformational forms and use them as the basis for examining molecular models for the activation, slow inactivation, and recovery processes. We compare structural similarities and differences in the pore domains, specifically in the transmembrane helices, the constrictions within the pore cavity, the activation gate at the cytoplasmic end of the last transmembrane helix, the C-terminal domain, and the selectivity filter. We discuss the observed differences in the context of previous models for opening, closing, and inactivation, and present a new structure-based model for the functional transitions. Our proposed prokaryotic channel activation mechanism is then compared with the activation transition in eukaryotic sodium channels

Distinctive Role of KV1.1 Subunits in the Biology and Functions of Low Threshold K+ Channels with Implication for Neurological Disease

This document is the Accepted Manuscript version of the following article: Saak V. Ovsepian; Marie LeBerre; Volker Steuber; Valerie B. O’Leary; Christian Leibold; & J. Oliver Dolly; ‘Distinctive role of KV1.1 subunit in the biology and functions of low threshold K+ channels with implications for neurological disease’, Pharmacology & Therapeutics, Vol. 159, March 2016, pp. 93-101. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ The version of record is available on line at doi: http:dx.doi.org/10.1016/j.pharmthera.2016.01.005 © 2016 Elsevier Inc. All rights reserved.The diversity of pore-forming subunits of KV1 channels (KV1.1–KV1.8) affords their physiological versatility and predicts a range of functional impairments resulting from genetic aberrations. Curiously, identified so far human neurological conditions associated with dysfunctions of KV1 channels have been linked exclusively to mutations in the KCNA1 gene encoding for the KV1.1 subunit. The absence of phenotypes related to irregularities in other subunits, including the prevalent KV1.2 subunit of neurons is highly perplexing given that deletion of the corresponding kcna2 gene in mouse models precipitates symptoms reminiscent to those of KV1.1 knockouts. Herein, we critically evaluate the molecular and biophysical characteristics of the KV1.1 protein in comparison with others and discuss their role in the greater penetrance of KCNA1 mutations in humans leading to the neurological signs of episodic ataxia type 1 (EA1). Future research and interpretation of emerging data should afford new insights towards a better understanding of the role of KV1.1 in integrative mechanisms of neurons and synaptic functions under normal and disease conditionsPeer reviewedFinal Accepted Versio

Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding