Roles In Electrical Signaling Research Articles

Identification of sodium channel toxins from marine cone snails of the subgenera Textilia and Afonsoconus

Voltage-gated sodium (NaV) channels are transmembrane proteins that play a critical role in electrical signaling in the nervous system and other excitable tissues. µ-Conotoxins are peptide toxins from the venoms of marine cone snails (genus Conus) that block NaV channels with nanomolar potency. Most species of the subgenera Textilia and Afonsoconus are difficult to acquire; therefore, their venoms have yet to be comprehensively interrogated for µ-conotoxins. The goal of this study was to find new µ-conotoxins from species of the subgenera Textilia and Afonsoconus and investigate their selectivity at human NaV channels. Using RNA-seq of the venom gland of Conus (Textilia) bullatus, we identified 12 µ-conotoxin (or µ-conotoxin-like) sequences. Based on these sequences we designed primers which we used to identify additional µ-conotoxin sequences from DNA extracted from historical specimens of species from Textilia and Afonsoconus. We synthesized six of these µ-conotoxins and tested their activity on human NaV1.1–NaV1.8. Five of the six synthetic peptides were potent blockers of human NaV channels. Of these, two peptides (BuIIIB and BuIIIE) were potent blockers of hNaV1.3. Three of the peptides (BuIIIB, BuIIIE and AdIIIA) had submicromolar activity at hNaV1.7. This study serves as an example of the identification of new peptide toxins from historical DNA and provides new insights into structure–activity relationships of µ-conotoxins with activity at hNaV1.3 and hNaV1.7.

Modulation of Pore Opening of Eukaryotic Sodium Channels by π-Helices in S6.

Voltage-gated sodium channels are heterotetrameric sodium selective ion channels that play a central role in electrical signaling in excitable cells. With recent advances in structural biology, structures of eukaryotic sodium channels have been captured in several distinct conformations corresponding to different functional states. The secondary structure of the pore lining S6 helices of subunits DI, DII, and DIV has been captured with both short π-helix stretches and in fully α-helical conformations. The relevance of these secondary structure elements for pore gating is not yet understood. Here, we propose that a π-helix in at least DI-S6, DIII-S6, and DIV-S6 results in a fully conductive state. On the other hand, the absence of π-helix in either DI-S6 or DIV-S6 yields a subconductance state, and its absence from both DI-S6 and DIV-S6 yields a nonconducting state. This work highlights the impact of the presence of a π-helix in the different S6 helices of an expanded pore on pore conductance, thus opening new doors toward reconstructing the entire conformational landscape along the functional cycle of Nav Channels and paving the way to the design of state-dependent modulators.

Ricca’s factors as mobile proteinaceous effectors of electrical signaling

Leaf-feeding insects trigger high-amplitude, defense-inducing electrical signals called slow wave potentials (SWPs). These signals are thought to be triggered by the long-distance transport of low molecular mass elicitors termed Ricca's factors. We sought mediators of leaf-to-leaf electrical signaling in Arabidopsis thaliana and identified them as β-THIOGLUCOSIDE GLUCOHYDROLASE 1 and 2 (TGG1 and TGG2). SWP propagation from insect feeding sites was strongly attenuated in tgg1 tgg2 mutants and wound-response cytosolic Ca2+ increases were reduced in these plants. Recombinant TGG1 fed into the xylem elicited wild-type-like membrane depolarization and Ca2+ transients. Moreover, TGGs catalyze the deglucosidation of glucosinolates. Metabolite profiling revealed rapid wound-induced breakdown of aliphatic glucosinolates in primary veins. Using invivo chemical trapping, we found evidence for roles of short-lived aglycone intermediates generated by glucosinolate hydrolysis in SWP membrane depolarization. Our findings reveal a mechanism whereby organ-to-organ protein transport plays a major role in electrical signaling.

An α-π transition in S6 shapes the conformational cycle of the bacterial sodium channel NavAb.

Voltage-gated sodium channels play an important role in electrical signaling in excitable cells. In response to changes in membrane potential, they cycle between nonconducting and conducting conformations. With recent advances in structural biology, structures of sodium channels have been captured in several distinct conformations, which are thought to represent different functional states. However, it has been difficult to capture the intrinsically transient open state. We recently showed that a proposed open state of the bacterial sodium channel NavMs was not conductive and that a conformational change involving a transition to a π-helix in the pore-lining S6 helix converted this structure into a conducting state. However, the relevance of this structural feature in other sodium channels, and its implications for the broader gating cycle, remained unclear. Here, we propose a comparable open state of another class of bacterial channel from Aliarcobacter butzleri (NavAb) with characteristic pore hydration, ion permeation, and drug binding properties. Furthermore, we show that a π-helix transition can lead to pore opening and that such a conformational change blocks fenestrations in the inner helix bundle. We also discover that a region in the C-terminal domain can undergo a disordering transition proposed to be important for pore opening. These results support a role for a π-helix transition in the opening of NavAb, enabling new proposals for the structural annotation and drug modulation mechanisms in this important sodium channel model.

Unappreciated Roles for K+ Channels in Bacterial Physiology.

Potassium (K+) channels are highly conserved proteins found in all domains of life, that allow for selective movement of K+ ions across membranes. Despite their broad distribution, the physiological roles of individual members of this diverse channel family have only been thoroughly explored in eukaryotic systems, where they have critical functions in a variety of cellular processes. Recent studies have demonstrated that bacterial K+ channels have integral roles in electrical signaling, information propagation, and intercellular communication. We discuss how these novel findings impact our understanding of bacterial physiology and the need to continue to explore the native roles of ion channels in microbes.

Recombinant Expression and Refolding of a Potassium Channel-Activating Three-Finger Toxin

Tx7335 is a novel three-finger peptide toxin that was originally isolated from the Eastern Green Mamba venom. A unique characteristic of Tx7335 is the ability to increase duration and frequency of openings in the potassium channel KcsA. Potassium channels play an important role in electrical signaling of excitable cells. Dysfunction of the channels can lead to major health problems such as certain heart and autoimmune diseases. Potassium channels are natural targets for venomous animals seeking to paralyze their prey. As a result, toxins have been shown to be an effective method of modulating and studying potassium channels. To obtain sufficient amounts of Tx7335 for future studies into its mechanism of action, we are recombinantly expressing and purifying this toxin. A synthetic gene encoding for Tx7335 was cloned into an expression vector expressing the toxin as a thioredoxin fusion protein with a TEV protease cleavage site in between them. The fusion protein was overexpressed in E coli. BL21(DE3) C43 expression strain and purified by Nickel affinity chromatography. The toxin was released from the fusion protein by cleavage with TEV protease and refolded by addition of reduced and oxidized glutathione in 1 to 10 ratio. Tx7335 was further purified through Cation exchange chromatography. Refolding was assessed by mass spectrometry, and the recombinant toxin will be used for future functional and structural studies of toxin-KcsA interactions.

Direct Evidence for a Similar Molecular Mechanism Underlying Shaker Kv Channel Fast Inactivation and Clustering

The fast inactivation and clustering functions of voltage-dependent potassium channels play fundamental roles in electrical signaling. Recent evidence suggests that both these distinct channel functions rely on intrinsically disordered N- and C-terminal cytoplasmic segments that function as entropic clocks to time channel inactivation or scaffold protein-mediated clustering, both relying on what can be described as a “ball and chain” binding mechanism. Although the mechanisms employed in each case are seemingly analogous, both were put forward based on bulky chain deletions and further exhibit differences in reaction order. These considerations raised the question of whether the molecular mechanisms underlying Kv channel fast inactivation and clustering are indeed analogous. By taking a “chain”-level chimeric channel approach involving long and short spliced inactivation or clustering “chain” variants of the Shaker Kv channel, we demonstrate the ability of native inactivation and clustering “chains” to substitute for each other in a length-dependent manner, as predicted by the “ball and chain” mechanism. Our results thus provide direct evidence arguing that the two completely unrelated Shaker Kv channel processes of fast inactivation and clustering indeed occur according to a similar molecular mechanism.

Interpreting the functional role of a novel interaction motif in prokaryotic sodium channels.

Voltage-gated sodium channels enable the translocation of sodium ions across cell membranes and play crucial roles in electrical signaling by initiating the action potential. In humans, mutations in sodium channels give rise to several neurological and cardiovascular diseases, and hence they are targets for pharmaceutical drug developments. Prokaryotic sodium channel crystal structures have provided detailed views of sodium channels, which by homology have suggested potentially important functionally related structural features in human sodium channels. A new crystal structure of a full-length prokaryotic channel, NavMs, in a conformation we proposed to represent the open, activated state, has revealed a novel interaction motif associated with channel opening. This motif is associated with disease when mutated in human sodium channels and plays an important and dynamic role in our new model for channel activation.

Modulation of a Bacterial Voltage-Gated Sodium Channel by the Anti-Epileptic Drug Lacosamide

Voltage-gated sodium channels play essential roles in electrical signaling and are primary targets in the treatment of pathologies such as epilepsy. Bacterial sodium channels are known to exhibit similar modulation by anti-epileptic agents, yet the interactions and modulation mechanisms remain unsolved at the molecular level. We have carried out multi-microsecond simulations of the bacterial NavAb channel in the presence of the drug lacosamide, a novel and effective antiepileptic drug which appears to favor slow inactivated states. We have observed drug access to the channel pore via membrane-bound fenestrations, binding to residue F203 that is a surrogate for the highly conserved FS6 in mammalian channels. We also see significant binding at the cytoplasmic gate (N211), as well as interactions with charged Arg side chains throughout entire voltage sensor domains. Calculated dissociation constants reveal that lacosamide has stronger affinity in specific conformations of the pore-forming S6 helices associated with slow inactivation and consistent with experimental evidence from mammalian channels (1). Moreover, we hypothesize that asymmetric binding within the tetrameric channel may instigate pore collapse, helping to explain this state-dependence. These studies provide new insight into sodium channel modulation, and predictions to assist in the development of improved anti-epileptic drugs.1. Errington et al (2008) Mol. Pharm., 73(1), 157-169

Sodium Selective Conduction, Inactivation and Inhibition Mechanisms using the Bacterial NavAb Channel

Voltage-gated sodium channels play essential roles in electrical signaling in the nervous system and are key pharmacological targets. We have carried out multi-microsecond Anton simulations of the bacterial NavAb channel to reveal ion conduction intimately connected with conformational fluctuations of the protein pore (C Boiteux, I Vorobyov & TW Allen, 2014 PNAS 111:3454), and a multiple-ion mechanism underlying Na+ versus K+ selectivity. We observe collapse of the pore domain, involving residues identified in pore-based slow inactivation and drug binding in prokaryotic and eukaryotic channels. We demonstrate high affinity binding of anti-epileptic and local anesthetic drugs to F203, that is a surrogate for the highly conserved FS6 in mammalian sodium channels, as well as low affinity sites with potential roles in channel inhibition. We observe two drug access pathways, including a previously suggested lipophilic route via membrane-bound fenestrations, and an aqueous pathway through the channel pore, despite being closed (C Boiteux, et al. 2014 PNAS 111:13057). These studies provide new insight into Nav function and modulation, and predictions to assist future drug development.

Molecular biology of insect sodium channels and pyrethroid resistance

Voltage-gated sodium channels are essential for the initiation and propagation of the action potential in neurons and other excitable cells. Because of their critical roles in electrical signaling, sodium channels are targets of a variety of naturally occurring and synthetic neurotoxins, including several classes of insecticides. This review is intended to provide an update on the molecular biology of insect sodium channels and the molecular mechanism of pyrethroid resistance. Although mammalian and insect sodium channels share fundamental topological and functional properties, most insect species carry only one sodium channel gene, compared to multiple sodium channel genes found in each mammalian species. Recent studies showed that two posttranscriptional mechanisms, alternative splicing and RNA editing, are involved in generating functional diversity of sodium channels in insects. More than 50 sodium channel mutations have been identified to be responsible for or associated with knockdown resistance (kdr) to pyrethroids in various arthropod pests and disease vectors. Elucidation of molecular mechanism of kdr led to the identification of dual receptor sites of pyrethroids on insect sodium channels. Many of the kdr mutations appear to be located within or close to the two receptor sites. The accumulating knowledge of insect sodium channels and their interactions with insecticides provides a foundation for understanding the neurophysiology of sodium channels in vivo and the development of new and safer insecticides for effective control of arthropod pests and human disease vectors.

Uncovering the Links Between Conformational Flexibility and Function for a Bacterial Voltage-Gated Sodium Channel

Voltage-gated sodium channels play essential roles in electrical signaling in the nervous system and are key pharmacological targets for a range of disorders. We have carried out fully-atomistic simulations on the multi-microsecond timescale to investigate sodium channel function. These simulations have produced complete unbiased free energy maps that reveal complex multi-ion conduction mechanisms for sodium (as well as potassium and calcium ions). They have also uncovered a surprising level of conformational flexibility of the channel, including concerted movements of the selectivity filter's EEEE ring sequence side chains, and significant changes in pore domain structure as a function of glutamate protonation states. We have observed asymmetrical rearrangements of the activation gate, resembling previously proposed inactivated structures, as well as helix bending involving residues critical for slow inactivation. We report how these structural changes regulate access to lipid-facing fenestrations and the binding of the local anesthetic and anticonvulsant drugs phenytoin and benzocaine, providing new insight into the molecular mechanisms of sodium channel inhibition.

Trafficking Defective Mutations Modulate NaV1.5 N—Glycosylation States

Voltage gated sodium channels are membrane proteins that play a critical role in electrical signaling of excitable cells. Amongst this family, the isoform Nav1.5 is responsible for the initiation and propagation of cardiac action potentials. As most of membrane proteins, Nav1.5 is well known to be a glycoprotein with ∼5% of its total weight corresponding to carbohydrates. To date, it has been shown that Nav1.5 glycosylations such as sialylations influence biophysical properties of this channel protein.

Inter-Subunit Interactions across the Upper Voltage Sensing-Pore Domain Interface Contribute to the Concerted Pore Opening Transition of Kv Channels

The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

The role of electrical signals in murine corneal wound re‐epithelialization

Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields (EF) to promote wound healing is the basis of Medicare-approved electric stimulation therapy. This study investigated the roles for EFs in wound re-epithelialization, using the Pax6(+/-) mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild-type (WT) and Pax6(+/-) corneal epithelial cells showed increased migration speeds in response to applied EFs in vitro. However, only Pax6(+/+) cells demonstrated consistent directional galvanotaxis towards the cathode, with activation of pSrc signaling, polarized to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6(+/-) corneas exhibited reversed endogenous wound-induced currents (the wound was an anode). These corneas healed at the same rate as WT. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for WT or mutant corneas. Furthermore, during healing in vivo, no polarization of pSrc was observed. We found little evidence that Src-dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous EFs do not drive long-term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signaling initiation.

Functional KCNH1 Potassium Channels in Danio Rerio are Essential for Early Development

The physiological role of KCNH1 channels has not been clarified in detail yet, but the predominant neuronal expression in mammals indicates a role in electrical signaling. The only described physiological function of KCNH1 so far is the promotion of myoblast fusion. Moreover, KCNH1 channels apparently enhance proliferation of cancer cells, thus exhibiting oncogenic potential. Many genes involved in cancerogenesis play a physiological role during embryonic development. To study KCNH1 channel function in this respect, we surveyed genomic databases of Danio rerio, a widely used vertebrate-development model organism, and found two putative kcnh1 paralogs located on chromosomes 17 (kcnh1a) and 22 (kcnh1b).

Ether ℵ go-go Potassium Channels KCNH1 and KCNH5 Have Four Functional Orthologs in Danio Rerio

The human ether a go-go channels KCNH1 and KCNH5 form a subgroup with unique functional properties within the eight-membered KCNH family of voltage-gated potassium channels. In mammals, the expression of both genes is mainly restricted to the brain, implying a role in electrical signaling of neurons. Heterologous expression in mammalian cells and Xenopus oocytes revealed that ether a go-go channels are sensitive to intracellular Ca2+/calmodulin. The second functional characteristic of both channels is a pronounced slowing of activation kinetics upon binding of extracellular divalent cations. To elucidate the physiological relevance of such properties, an adequate model organism would be desirable. Here we studied the expression of ether a go-go orthologs in zebrafish. Due to a whole-genome duplication during evolution of teleost fish, mammalian genes can have two functional orthologs in zebrafish. However, for the majority of duplicated genes, one gene copy is lost or present as nonfunctional pseudogene only. Using in silico screening of genome databases and cloning from reverse transcribed mRNA, we could show that for each of the two human ether a go-go channels two functional orthologs exist in zebrafish. Upon expression in Xenopus oocytes, all four genes generated functional channels with current-voltage relationships similar to the human orthologs, characterized by a very negative threshold of the activation voltages. A more pronounced slowing of activation kinetics in the presence of extracellular Mg2+ ions clearly distinguished KCNH5 and its two fish orthologs from human KCNH1 and the corresponding fish channels. In summary, the structural and functional conservation between human and fish ether a go-go channels makes zebrafish a promising model, but gene duplication must be taken into account. This may also be relevant for the closely related paralogs ERG and ELK.

Inverse Modes of Coupling in Leak and Voltage-activated K+ Channel Pore Gates Underlie their Distinct Roles in Electrical Signaling

Voltage-activated (Kv) and leak (K2P) potassium channels play key, yet distinct roles in electrical signaling in the nervous system. Here, we examined how differences in the operation of the activation and slow inactivation pore gates of Kv and K2P channels underlie their unique roles in electrical signaling. We report that (1) leak potassium channels possess a lower activation gate, (2) the activation gate is an important determinant controlling the conformational stability of the K+ channel pore, (3) the lower activation and upper slow inactivation gates of leak channels cross-talk and (4) in contrast to Kv channels, where the two pore gates are negatively-coupled these two gates are positively-coupled in K2P channels. Our results thus demonstrate how basic thermodynamic properties of the K+ channel pore, particularly conformational stability and coupling between the pore gates, underlie the specialized roles of Kv and K2P channel families in electrical signaling.

Inverse coupling in leak and voltage-activated K+ channel gates underlies distinct roles in electrical signaling

Voltage-activated (Kv) and leak (K(2P)) K(+) channels have key, yet distinct, roles in electrical signaling in the nervous system. Here we examine how differences in the operation of the activation and slow inactivation pore gates of Kv and K(2P) channels underlie their unique roles in electrical signaling. We report that (i) leak K(+) channels possess a lower activation gate, (ii) the activation gate is an important determinant controlling the conformational stability of the K(+) channel pore, (iii) the lower activation and upper slow inactivation gates of leak channels cross-talk and (iv) unlike Kv channels, where the two gates are negatively coupled, these two gates are positively coupled in K(2P) channels. Our results demonstrate how basic thermodynamic properties of the K(+) channel pore, particularly conformational stability and coupling between gates, underlie the specialized roles of Kv and K(2P) channel families in electrical signaling.

Cardiovascular KCNQ (Kv7) Potassium Channels: Physiological Regulators and New Targets for Therapeutic Intervention

Potassium channels play an important role in electrical signaling of excitable cells such as neurons, cardiac myocytes, and vascular smooth muscle cells (VSMCs). In particular, the KCNQ (Kv7) family of voltage-activated K(+) channels functions to stabilize negative resting membrane potentials and thereby opposes electrical excitability. Of the five known members of the mammalian Kv7 family, Kv7.1 was originally recognized for its role in cardiac myocytes, where it contributes to repolarization of the cardiac action potential. Kv7.2 to Kv7.5 were first discovered in neurons, in which they play a well characterized role in neurotransmitter-stimulated action potential firing. Over the past 5 years, important new roles for Kv7 channels have been identified. Kv7 channels have been found to be expressed in VSMCs from several vascular beds where they contribute to the regulation of vascular tone. There is evidence that Kv7.5 channels in VSMCs are targeted by the hormone vasopressin to mediate its physiological vasoconstrictor actions and evidence that neuronal Kv7 channels in the baroreceptors of the aortic arch adjust the sensitivity of the mechanosensitive neurons to changes in arterial blood pressure. These newly identified physiological roles for Kv7 channels in the cardiovascular system warrant increased attention because pharmacological modulators of this family of channels are being used clinically to treat a variety of neurological disorders. This raises questions about the cardiovascular side effects associated with existing therapies, but there is also obvious potential to capitalize on the established and evolving pharmacology of these channels to develop new therapies for cardiovascular diseases.