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

Abstract

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.

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Figure 1: Structure and function of the CaVAb channel.
Figure 2: Ca2+-binding sites in and near the selectivity filter of NaVAb, CaVAb and their derivatives.
Figure 3: Ion binding and block of CaVAb and its derivatives.
Figure 4: Catalytic cycle for Ca2+ conductance by CaVAb.

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Accessions

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes: 4MS2 (TLDDWSN, 15 mM Ca2+), 4MTF (TLDDWSN, 0.5 mM Ca2+), 4MTG (TLDDWSN, 2.5 mM Ca2+), 4MTO (TLDDWSN, 5 mM Ca2+), 4MVM (TLDDWSN, 10 mM Ca2+), 4MVO (TLDDWSN, 15 mM Ca2+), 4MVQ (TLDDWSD, 15 mM Ca2+), 4MVR (TLDDWSD, 100 mM Mn2+), 4MVS (TLDDWSD, 100 mM Cd2+), 4MVZ (TLEDWSD, 15 mM Ca2+), 4MW3 (TLDDWSM, 15 mM Ca2+), 4MVU (TLEDWSM, 15 mM Ca2+), 4MW8 (NavAb, 15 mM Ca2+).

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Acknowledgements

We are grateful to the beamline staff at the Advanced Light Source (BL8.2.1 and BL8.2.2) for their assistance during data collection. Research reported in this publication was supported by the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH) under award number R01NS015751 (W.A.C.), the National Heart, Lung, and Blood Institute (NHLBI) of the NIH under award number R01HL112808 (W.A.C. and N.Z.) and a National Research Service Award from training grant T32GM008268 (T.M.H.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This work was also supported by the Howard Hughes Medical Institute (N.Z.).

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L.T., T.M.G.E.-D., J.P., T.S., N.Z. and W.A.C. designed the experiments. J.P. initiated the experimental work. L.T. conducted the protein purification, crystallization and diffraction experiments. L.T., J.P. and N.Z. determined and analysed the structures of the apo and cation-bound forms of CaVAb and the intermediate CaVAb constructs. T.M.G.E.-D. and T.S. performed physiological studies of CaVAb and related constructs. G.Q.M. and T.M.H. made the constructs and performed the preliminary data collection. All authors interpreted the structures in light of the physiological data. L.T., N.Z. and W.A.C. wrote the manuscript with input from all co-authors. W.A.C. and N.Z. are co-senior authors.

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Correspondence to Ning Zheng or William A. Catterall.

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Tang, L., Gamal El-Din, T., Payandeh, J. et al. Structural basis for Ca2+ selectivity of a voltage-gated calcium channel. Nature 505, 56–61 (2014). https://doi.org/10.1038/nature12775

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