Modeling of the Outer Vestibule and Selectivity Filter of the L-Type Ca2+ Channel

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Abstract
Using the KcsA bacterial K+ channel crystal structure [Doyle, D. A., et al. (1998) Science 280, 69−74] and the model of the outer vestibule of the Na+ channel [Lipkind, G. M., and Fozzard, H. A. (2000) Biochemistry 39, 8161−8170] as structural templates, we propose a structural model of the outer vestibule and selectivity filter of the pore of the Ca2+ channel (α1C or Cav1.2). The Ca2+ channel P loops were modeled by α-helix−turn−β-strand motifs, with the glutamate residues of the EEEE motif located in the turns. P loops were docked in the extracellular part of the inverted teepee structure formed by S5 and S6 α-helices with backbone coordinates from the M1 and M2 helices of the KcsA crystal structure. This construction results in a conical outer vestibule that tapers to the selectivity filter at the bottom. The modeled selectivity ring forms a wide open pore (∼6 Å) in the absence of Ca2+. When Ca2+ is present (∼1 μM), all four glutamate side chains move to the center and form a cage around the dehydrated Ca2+ ion, blocking the pore. In the millimolar concentration range, Ca2+ also interacts with two low-affinity sites located externally and internally, which were modeled by the same carboxylate groups of the selectivity filter. Calculation of the resulting electrostatic potentials show that the single Ca2+ ion is located in an electrostatic trap. Only when three Ca2+ ions are bound simultaneously in the high- and low-affinity sites of the selectivity filter is Ca2+ able to overcome electrostatic attraction, permitting Ca2+ flux.