Voltage-gated sodium (Nav) channels are essential for the rapid depolarization of nerve and muscle1, and are important drug targets2. A family of bacterial Nav channels, exemplified by NaChBac (Na+-selective Channel of Bacteria)3, provides a good model system for structure-function analysis. Here we report the crystal structure of NavAP, a NaChBac orthologue from marine bacteria alpha proteobacterium HIMB114, at 3.05 Å resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr178 and Leu179 constitute an inner site within the selectivity filter (178TLSSWE183) where a Ca2+ can bind and resides in the crystal structure. The outer mouth of the Na+ selectivity filter, defined by Ser181 and Glu183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation with all the gating charges exposing to the extracellular side. We hypothesize that NavAP is captured in an inactivated conformation. Comparison of NavAP with NavAb4 reveals significant conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.

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Voltage-gated calcium (CaV) channels catalyze rapid, highly selective influx of Ca2+ into cells despite 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 play 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 mechanisms of ion selectivity and conductance by vertebrate CaV channels.

(R)-Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B 6 . These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.