Structural basis for Ca(2+) selectivity of a voltage-gated calcium channel

Voltage-gated calcium (Ca(V)) channels catalyze rapid, highly selective influx of Ca(2+) into cells despite 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(V)Ab. Our results reveal interactions of hydrated Ca(2+) with two high-affinity Ca(2+)-binding sites followed by a third lower-affinity site that would coordinate Ca(2+) as it moves inward. At the selectivity filter entry, Site 1 is formed by four carboxyl side-chains, which play a critical role in determining Ca(2+) selectivity. Four carboxyls plus four backbone carbonyls form Site 2, which is targeted by the blocking cations, Cd(2+) and Mn(2+), 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 Ca(2+) 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(V)Ab model establishes a structural framework for understanding mechanisms of ion selectivity and conductance by vertebrate Ca(V) channels.