Voltage-gated calcium (Ca_V) channels catalyse rapid, highly selective influx of Ca²⁺ into cells despite a 70-fold higher extracellular concentration of Na⁺. How Ca_V 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 Na_V channel Na_VAb. 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 Ca_VAb model establishes a structural framework for understanding the mechanisms of ion selectivity and conductance by vertebrate Ca_V channels.

Significance: Bacterial voltage-gated sodium channels serve as models of their vertebrate counterparts because they have similar functional components in a simpler structure. We present high-resolution structures of tightly closed and open states. In the closed state, the activation gate fully occludes the conduction pathway, and the intracellular C-terminal domain is revealed as a long four-helix bundle. In the open state, the activation gate has an orifice of ∼10 Å. Molecular dynamics simulations confirm that this conformation would allow permeation of hydrated Na⁺. These structures are significant advances because they provide a complete 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.

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 Na _V Ab, 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 Na _v Ab, 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.