Dual receptor-sites reveal the structural basis for hyperactivation of sodium channels by poison-dart toxin batrachotoxin.

The poison dart toxin batrachotoxin is exceptional for its high potency and toxicity, and for its multifaceted modification of the function of voltage-gated sodium channels. By using cryogenic electron microscopy, we identify two homologous, but nonidentical receptor sites that simultaneously bind two molecules of toxin, one at the interface between Domains I and IV, and the other at the interface between Domains III and IV of the cardiac sodium channel. Together, these two bound toxin molecules stabilize α/π helical conformation in the S6 segments that gate the pore, and one of the bound BTX-B molecules interacts with the crucial Lys1421 residue that is essential for sodium conductance and selectivity via an apparent water-bridged hydrogen bond. Overall, our structure provides insight into batrachotoxin's potency, efficacy, and multifaceted functional effects on voltage-gated sodium channels via a dual receptor site mechanism.

Fenestropathy of Voltage-Gated Sodium Channels.

Voltage-gated sodium channels (Nav) are responsible for the initiation and propagation of action potentials in excitable cells. From pain to heartbeat, these integral membrane proteins are the ignition stations for every sensation and action in human bodies. They are large (>200 kDa, 24 transmembrane helices) multi-domain proteins that couple changes in membrane voltage to the gating cycle of the sodium-selective pore. Nav mutations lead to a multitude of diseases - including chronic pain, cardiac arrhythmia, muscle illnesses, and seizure disorders - and a wide variety of currently used therapeutics block Nav. Despite this, the mechanisms of action of Nav blocking drugs are only modestly understood at this time and many questions remain to be answered regarding their state- and voltage-dependence, as well as the role of the hydrophobic membrane access pathways, or fenestrations, in drug ingress or egress. Nav fenestrations, which are pathways that connect the plasma membrane to the central cavity in the pore domain, were discovered through functional studies more than 40 years ago and once thought to be simple pathways. A variety of recent genetic, structural, and pharmacological data, however, shows that these fenestrations are actually key functional regions of Nav that modulate drug binding, lipid binding, and influence gating behaviors. We discovered that some of the disease mutations that cause arrhythmias alter amino acid residues that line the fenestrations of Nav1.5. This indicates that fenestrations may play a critical role in channel's gating, and that individual genetic variation may also influence drug access through the fenestrations for resting/inactivated state block. In this review, we will discuss the channelopathies associated with these fenestrations, which we collectively name "Fenestropathy," and how changes in the fenestrations associated with the opening of the intracellular gate could modulate the state-dependent ingress and egress of drugs binding in the central cavity of voltage gated sodium channels.

Open-state structure and pore gating mechanism of the cardiac sodium channel.

The heartbeat is initiated by voltage-gated sodium channel NaV1.5, which opens rapidly and triggers the cardiac action potential; however, the structural basis for pore opening remains unknown. Here, we blocked fast inactivation with a mutation and captured the elusive open-state structure. The fast inactivation gate moves away from its receptor, allowing asymmetric opening of pore-lining S6 segments, which bend and rotate at their intracellular ends to dilate the activation gate to ∼10 Å diameter. Molecular dynamics analyses predict physiological rates of Na+ conductance. The open-state pore blocker propafenone binds in a high-affinity pose, and drug-access pathways are revealed through the open activation gate and fenestrations. Comparison with mutagenesis results provides a structural map of arrhythmia mutations that target the activation and fast inactivation gates. These results give atomic-level insights into molecular events that underlie generation of the action potential, open-state drug block, and fast inactivation of cardiac sodium channels, which initiate the heartbeat.

Structure and Pharmacology of Voltage-Gated Sodium and Calcium Channels.

Voltage-gated sodium and calcium channels are evolutionarily related transmembrane signaling proteins that initiate action potentials, neurotransmission, excitation-contraction coupling, and other physiological processes. Genetic or acquired dysfunction of these proteins causes numerous diseases, termed channelopathies, and sodium and calcium channels are the molecular targets for several major classes of drugs. Recent advances in the structural biology of these proteins using X-ray crystallography and cryo-electron microscopy have given new insights into the molecular basis for their function and pharmacology. Here we review this recent literature and integrate findings on sodium and calcium channels to reveal the structural basis for their voltage-dependent activation, fast and slow inactivation, ion conductance and selectivity, and complex pharmacology at the atomic level. We conclude with the theme that new understanding of the diseases and therapeutics of these channels will be derived from application of the emerging structural principles from these recent structural analyses.

Structure of the Cardiac Sodium Channel.

Voltage-gated sodium channel Nav1.5 generates cardiac action potentials and initiates the heartbeat. Here, we report structures of NaV1.5 at 3.2-3.5 Å resolution. NaV1.5 is distinguished from other sodium channels by a unique glycosyl moiety and loss of disulfide-bonding capability at the NaVß subunit-interaction sites. The antiarrhythmic drug flecainide specifically targets the central cavity of the pore. The voltage sensors are partially activated, and the fast-inactivation gate is partially closed. Activation of the voltage sensor of Domain III allows binding of the isoleucine-phenylalanine-methionine (IFM) motif to the inactivation-gate receptor. Asp and Ala, in the selectivity motif DEKA, line the walls of the ion-selectivity filter, whereas Glu and Lys are in positions to accept and release Na+ ions via a charge-delocalization network. Arrhythmia mutation sites undergo large translocations during gating, providing a potential mechanism for pathogenic effects. Our results provide detailed insights into Nav1.5 structure, pharmacology, activation, inactivation, ion selectivity, and arrhythmias.

Structural Basis for Diltiazem Block of a Voltage-Gated Ca2+ Channel.

Diltiazem is a widely prescribed Ca2+ antagonist drug for cardiac arrhythmia, hypertension, and angina pectoris. Using the ancestral CaV channel construct CaVAb as a molecular model for X-ray crystallographic analysis, we show here that diltiazem targets the central cavity of the voltage-gated Ca2+ channel underneath its selectivity filter and physically blocks ion conduction. The diltiazem-binding site overlaps with the receptor site for phenylalkylamine Ca2+ antagonist drugs such as verapamil. The dihydropyridine Ca2+ channel blocker amlodipine binds at a distinct site and allosterically modulates the binding sites for diltiazem and Ca2+ Our studies resolve two distinct binding poses for diltiazem in the absence and presence of amlodipine. The binding pose in the presence of amlodipine may mimic a high-affinity binding configuration induced by voltage-dependent inactivation, which is favored by dihydropyridine binding. In this binding pose, the tertiary amino group of diltiazem projects upward into the inner end of the ion selectivity filter, interacts with ion coordination Site 3 formed by the backbone carbonyls of T175, and alters binding of Ca2+ to ion coordination Sites 1 and 2. Altogether, our results define the receptor site for diltiazem and elucidate the mechanisms for pore block and allosteric modulation by other Ca2+ channel-blocking drugs at the atomic level. SIGNIFICANCE STATEMENT: Calcium antagonist drugs that block voltage-gated calcium channels in heart and vascular smooth muscle are widely used in the treatment of cardiovascular diseases. Our results reveal the chemical details of diltiazem binding in a blocking position in the pore of a model calcium channel and show that binding of another calcium antagonist drug alters binding of diltiazem and calcium. This structural information defines the mechanism of drug action at the atomic level and provides a molecular template for future drug discovery.

Molecular dissection of multiphase inactivation of the bacterial sodium channel NaVAb.

Homotetrameric bacterial voltage-gated sodium channels share major biophysical features with their more complex eukaryotic counterparts, including a slow-inactivation mechanism that reduces ion-conductance activity during prolonged depolarization through conformational changes in the pore. The bacterial sodium channel NaVAb activates at very negative membrane potentials and inactivates through a multiphase slow-inactivation mechanism. Early voltage-dependent inactivation during one depolarization is followed by late use-dependent inactivation during repetitive depolarization. Mutations that change the molecular volume of Thr206 in the pore-lining S6 segment can enhance or strongly block early voltage-dependent inactivation, suggesting that this residue serves as a molecular hub controlling the coupling of activation to inactivation. In contrast, truncation of the C-terminal tail enhances the early phase of inactivation yet completely blocks late use-dependent inactivation. Determination of the structure of a C-terminal tail truncation mutant and molecular modeling of conformational changes at Thr206 and the S6 activation gate led to a two-step model of these gating processes. First, bending of the S6 segment, local protein interactions dependent on the size of Thr206, and exchange of hydrogen-bonding partners at the level of Thr206 trigger pore opening followed by the early phase of voltage-dependent inactivation. Thereafter, conformational changes in the C-terminal tail lead to late use-dependent inactivation. These results have important implications for the sequence of conformational changes that lead to multiphase inactivation of NaVAb and other sodium channels.

Fenestrations control resting-state block of a voltage-gated sodium channel.

Potency of drug action is usually determined by binding to a specific receptor site on target proteins. In contrast to this conventional paradigm, we show here that potency of local anesthetics (LAs) and antiarrhythmic drugs (AADs) that block sodium channels is controlled by fenestrations that allow drug access to the receptor site directly from the membrane phase. Voltage-gated sodium channels initiate action potentials in nerve and cardiac muscle, where their hyperactivity causes pain and cardiac arrhythmia, respectively. LAs and AADs selectively block sodium channels in rapidly firing nerve and muscle cells to relieve these conditions. The structure of the ancestral bacterial sodium channel NaVAb, which is also blocked by LAs and AADs, revealed fenestrations connecting the lipid phase of the membrane to the central cavity of the pore. We cocrystallized lidocaine and flecainide with NavAb, which revealed strong drug-dependent electron density in the central cavity of the pore. Mutation of the contact residue T206 greatly reduced drug potency, confirming this site as the receptor for LAs and AADs. Strikingly, mutations of the fenestration cap residue F203 changed fenestration size and had graded effects on resting-state block by flecainide, lidocaine, and benzocaine, the potencies of which were altered from 51- to 2.6-fold in order of their molecular size. These results show that conserved fenestrations in the pores of sodium channels are crucial pharmacologically and determine the level of resting-state block by widely used drugs. Fine-tuning drug access through fenestrations provides an unexpected avenue for structure-based design of ion-channel-blocking drugs.

Structural and Functional Analysis of Sodium Channels Viewed from an Evolutionary Perspective.

Voltage-gated sodium channels initiate and propagate action potentials in excitable cells. They respond to membrane depolarization through opening, followed by fast inactivation that terminates the sodium current. This ON-OFF behavior of voltage-gated sodium channels underlays the coding of information and its transmission from one location in the nervous system to another. In this review, we explore and compare structural and functional data from prokaryotic and eukaryotic channels to infer the effects of evolution on sodium channel structure and function.

Structures of closed and open states of a voltage-gated sodium channel.

Bacterial voltage-gated sodium channels (BacNavs) serve as models of their vertebrate counterparts. BacNavs contain conserved voltage-sensing and pore-forming domains, but they are homotetramers of four identical subunits, rather than pseudotetramers of four homologous domains. Here, we present structures of two NaVAb mutants that capture tightly closed and open states at a resolution of 2.8-3.2 Å. Introduction of two humanizing mutations in the S6 segment (NaVAb/FY: T206F and V213Y) generates a persistently closed form of the activation gate in which the intracellular ends of the four S6 segments are drawn tightly together to block ion permeation completely. This construct also revealed the complete structure of the four-helix bundle that forms the C-terminal domain. In contrast, truncation of the C-terminal 40 residues in NavAb/1-226 captures the activation gate in an open conformation, revealing the open state of a BacNav with intact voltage sensors. Comparing these structures illustrates the full range of motion of the activation gate, from closed with its orifice fully occluded to open with an orifice of ∼10 Å. Molecular dynamics and free-energy simulations confirm designation of NaVAb/1-226 as an open state that allows permeation of hydrated Na+, and these results also support a hydrophobic gating mechanism for control of ion permeation. These two structures allow completion of a closed-open-inactivated conformational cycle in a single voltage-gated sodium channel and give insight into the structural basis for state-dependent binding of sodium channel-blocking drugs.

Primary Care of the Patient with Asthma.

Obstructive lung disease includes asthma and chronic obstructive pulmonary disease (COPD). Because a previous issue of Medical Clinics of North America (2012;96[4]) was devoted to COPD, this article focuses on asthma in adults, and addresses some topics about COPD not addressed previously. Asthma is a heterogeneous disease marked by variable airflow obstruction and bronchial hyperreactivity. Onset is most common in early childhood, although many people develop asthma later in life. Adult-onset asthma presents a particular challenge in the primary care clinic because of incomplete understanding of the disorder, underreporting of symptoms, underdiagnosis, inadequate treatment, and high rate of comorbidity.

Structures of KcsA in complex with symmetrical quaternary ammonium compounds reveal a hydrophobic binding site.

Potassium channels allow for the passive movement of potassium ions across the cell membrane and are instrumental in controlling the membrane potential in all cell types. Quaternary ammonium (QA) compounds block potassium channels and have long been used to study the functional and structural properties of these channels. Here we describe the interaction between three symmetrical hydrophobic QAs and the prokaryotic potassium channel KcsA. The structures demonstrate the presence of a hydrophobic pocket between the inner helices of KcsA and provide insight into the binding site and blocking mechanism of hydrophobic QAs. The structures also reveal a structurally hidden pathway between the central cavity and the outside membrane environment reminiscent of the lateral fenestration observed in sodium channels that can be accessed through small conformational changes in the pore wall. We propose that the hydrophobic binding pocket stabilizes the alkyl chains of long-chain QA molecules and may play a key role in hydrophobic drug binding in general.

The respiratory substrate rhodoquinol induces Q-cycle bypass reactions in the yeast cytochrome bc(1) complex: mechanistic and physiological implications.

The mitochondrial cytochrome bc(1) complex catalyzes the transfer of electrons from ubiquinol to cyt c while generating a proton motive force for ATP synthesis via the "Q-cycle" mechanism. Under certain conditions electron flow through the Q-cycle is blocked at the level of a reactive intermediate in the quinol oxidase site of the enzyme, resulting in "bypass reactions," some of which lead to superoxide production. Using analogs of the respiratory substrates ubiquinol-3 and rhodoquinol-3, we show that the relative rates of Q-cycle bypass reactions in the Saccharomyces cerevisiae cyt bc(1) complex are highly dependent by a factor of up to 100-fold on the properties of the substrate quinol. Our results suggest that the rate of Q-cycle bypass reactions is dependent on the steady state concentration of reactive intermediates produced at the quinol oxidase site of the enzyme. We conclude that normal operation of the Q-cycle requires a fairly narrow window of redox potentials with respect to the quinol substrate to allow normal turnover of the complex while preventing potentially damaging bypass reactions.

Structural basis of TEA blockade in a model potassium channel.

Potassium channels catalyze the selective transfer of potassium across the cell membrane and are essential for setting the resting potential in cells, controlling heart rate and modulating the firing pattern in neurons. Tetraethylammonium (TEA) blocks ion conduction through potassium channels in a voltage-dependent manner from both sides of the membrane. Here we show the structural basis of TEA blockade by cocrystallizing the prokaryotic potassium channel KcsA with two selective TEA analogs. TEA binding at both sites alters ion occupancy in the selectivity filter; these findings underlie the mutual destabilization and voltage-dependence of TEA blockade. We propose that TEA blocks potassium channels by acting as a potassium analog at the dehydration transition step during permeation.