Tamer M. Gamal El-Din

Crystal structure of a voltage-gated sodium channel in two potentially inactivated states

In excitable cells, voltage-gated sodium (NaV) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance. Inactivation is a hallmark of NaV channel function and is critical for control of membrane excitability, but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type NaVAb channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2 Å resolution. Compared to previous structures of NaVAb channels with cysteine mutations in the pore-lining S6 helices (ref. 4), the S6 helices and the intracellular activation gate have undergone significant rearrangements: one pair of S6 helices has collapsed towards the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type NaVAb models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues that are analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains have also shifted around the perimeter of the pore module in wild-type NaVAb, compared to the mutant channel, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in NaV channel gating and inactivation. These potential inactivated-state structures provide new insights into NaV channel gating and novel avenues to drug development and therapy for a range of debilitating NaV channelopathies.

Structural basis for Ca2+ selectivity of a voltage-gated calcium channel.

Voltage-gated calcium (CaV) channels catalyse rapid, highly selective influx of Ca2+ into cells despite a 70-fold higher extracellular concentration of Na+. How CaV channels solve this fundamental biophysical problem remains unclear. Here we report physiological and crystallographic analyses of a calcium selectivity filter constructed in the homotetrameric bacterial NaV channel NaVAb. Our results reveal interactions of hydrated Ca2+ with two high-affinity Ca2+-binding sites followed by a third lower-affinity site that would coordinate Ca2+ as it moves inward. At the selectivity filter entry, Site 1 is formed by four carboxyl side chains, which have a critical role in determining Ca2+ selectivity. Four carboxyls plus four backbone carbonyls form Site 2, which is targeted by the blocking cations Cd2+ and Mn2+, with single occupancy. The lower-affinity Site 3 is formed by four backbone carbonyls alone, which mediate exit into the central cavity. This pore architecture suggests a conduction pathway involving transitions between two main states with one or two hydrated Ca2+ ions bound in the selectivity filter and supports a ‘knock-off’ mechanism of ion permeation through a stepwise-binding process. The multi-ion selectivity filter of our CaVAb model establishes a structural framework for understanding the mechanisms of ion selectivity and conductance by vertebrate CaV channels.

Structural basis for inhibition of a voltage-gated Ca 2+ channel by Ca 2+ antagonist drugs

Ca2+ antagonist drugs are widely used in therapy of cardiovascular disorders. Three chemical classes of drugs bind to three separate, but allosterically interacting, receptor sites on CaV1.2 channels, the most prominent voltage-gated Ca2+ (CaV) channel type in myocytes in cardiac and vascular smooth muscle. The 1,4-dihydropyridines are used primarily for treatment of hypertension and angina pectoris and are thought to act as allosteric modulators of voltage-dependent Ca2+ channel activation, whereas phenylalkylamines and benzothiazepines are used primarily for treatment of cardiac arrhythmias and are thought to physically block the pore. The structural basis for the different binding, action, and therapeutic uses of these drugs remains unknown. Here we present crystallographic and functional analyses of drug binding to the bacterial homotetrameric model CaV channel CaVAb, which is inhibited by dihydropyridines and phenylalkylamines with nanomolar affinity in a state-dependent manner. The binding site for amlodipine and other dihydropyridines is located on the external, lipid-facing surface of the pore module, positioned at the interface of two subunits. Dihydropyridine binding allosterically induces an asymmetric conformation of the selectivity filter, in which partially dehydrated Ca2+ interacts directly with one subunit and blocks the pore. In contrast, the phenylalkylamine Br-verapamil binds in the central cavity of the pore on the intracellular side of the selectivity filter, physically blocking the ion-conducting pathway. Structure-based mutations of key amino-acid residues confirm drug binding at both sites. Our results define the structural basis for binding of dihydropyridines and phenylalkylamines at their distinct receptor sites on CaV channels and offer key insights into their fundamental mechanisms of action and differential therapeutic uses in cardiovascular diseases.

A gating charge interaction required for late slow inactivation of the bacterial sodium channel NavAb

Voltage-gated sodium channels undergo slow inactivation during repetitive depolarizations, which controls the frequency and duration of bursts of action potentials and prevents excitotoxic cell death. Although homotetrameric bacterial sodium channels lack the intracellular linker-connecting homologous domains III and IV that causes fast inactivation of eukaryotic sodium channels, they retain the molecular mechanism for slow inactivation. Here, we examine the functional properties and slow inactivation of the bacterial sodium channel NavAb expressed in insect cells under conditions used for structural studies. NavAb activates at very negative membrane potentials (V1/2 of approximately −98 mV), and it has both an early phase of slow inactivation that arises during single depolarizations and reverses rapidly, and a late use-dependent phase of slow inactivation that reverses very slowly. Mutation of Asn49 to Lys in the S2 segment in the extracellular negative cluster of the voltage sensor shifts the activation curve ∼75 mV to more positive potentials and abolishes the late phase of slow inactivation. The gating charge R3 interacts with Asn49 in the crystal structure of NavAb, and mutation of this residue to Cys causes a similar positive shift in the voltage dependence of activation and block of the late phase of slow inactivation as mutation N49K. Prolonged depolarizations that induce slow inactivation also cause hysteresis of gating charge movement, which results in a requirement for very negative membrane potentials to return gating charges to their resting state. Unexpectedly, the mutation N49K does not alter hysteresis of gating charge movement, even though it prevents the late phase of slow inactivation. Our results reveal an important molecular interaction between R3 in S4 and Asn49 in S2 that is crucial for voltage-dependent activation and for late slow inactivation of NavAb, and they introduce a NavAb mutant that enables detailed functional studies in parallel with structural analysis.

Tracking S4 movement by gating pore currents in the bacterial sodium channel NaChBac

Comparison of the kinetics and voltage dependence of gating pore current conducted by S4 gating charge mutants supports the sliding-helix model of voltage sensor function and elucidates the pathogenic mechanisms underlying periodic paralysis syndromes.

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.

Functional and Molecular Interaction of Phenylalkylamines and Dihydropyridines with the Model Calcium Channel CavAb

Mammalian voltage-gated calcium channels (mCavs) are molecular targets of phenylalkylamines (PAAs) and dihydropyridines (DHPs), which are widely used antiarrhythmic and antihypertension drugs that block mCavs in a frequency- and voltage-dependent manner. The two distinct receptor sites for these drugs in mCavs have been elucidated through extensive photoaffinity labeling and mutagenesis studies, revealing key residues along the S5 and S6 transmembrane segments of domains III and IV of mCavs (Hockerman et al., 1997). We have recently reported the crystal structure of CavAb, which is a highly calcium-selective mutant of the prokaryotic sodium channel NavAb (Tang et al., 2014). Even though CavAb is separated evolutionarily from mCavs by more than one billion years, we found that PAAs and DHPs interact with CavAb in a similar state-dependent manner. The PAAs tested have Kds in the range of 250 nM to 800 nM, and verapamil shows low-affinity block of the resting state and high-affinity use-dependent block of activated and/or inactivated states. Mutations of T206 in the center of segment S6 reduced PAA affinity by 20-fold. In the case of DHPs, nimodipine showed voltage-dependent block of activated CavAb with a Kd of 194 nM, but no resting-state block was observed in the tested concentration range. DHPs shift the voltage dependence of inactivation up to 100 mV negatively. This indicates that DHP interaction with CavAb is state-dependent and greatly favors the inactivated state, similar to mCavs. Mutation of I199 in the outer region of segment S6, which is critical for binding DHPs in mCavs, reduced binding affinity 40-fold. Our results reveal surprising similarity of drug action on CavAb and mCavs and thereby support use of CavAb as a molecular model to determine the three-dimensional structure of these important drug receptor sites.