Introduction

The Ca²⁺ selectivity of voltage-gated calcium (Ca_v) channels remained unclear. They were thought to use a ‘knock-off’ methanism which requires multiple ion-binding cores, but mutational analyses supported a single high-affinity Ca²⁺ binding site. This paradox is explained by the mechanism proposed in this article, based on the crystal structures of Ca_vAb channels.

Methods

In this study, the authors created several mutants of bacterial Na_v channel Na_vAb by site directed mutagenesis using QuickChange at the selectivity filter region¹. Some of these mutations changes the activity of the channel from allowing efflux of Na⁺ to allowing influx of Ca²⁺ to different extents (quantified by the relative permeability of Ca²⁺ against Na⁺). Baculovirus were used as the vector to infect Trichopulsia ni cells. Proteins were collected from insect cells, purified, concentrated to ~20mg ml^-1, and reconstituted into DMPC:CHAPSO bicelles (Figure 1). Crystals were grown in a hanging-drop vapour-diffusion format.

Figure 1: The DMPC:CHAPSO bicelle. Bicelles are disks with a planar region formed by long chain lipid phospholipids and a rim composed of short chain phospholipids or detergent molecules. It provides an environment that resembles a lipid bilayer where membrane proteins’ natively resides. Membrane proteins can be incorporated into bicelles for functional studies as well as crystallisation.

Before X-ray diffraction data collection, crystals were soaked with cryo-protectant solutions containing Ca²⁺, Mn²⁺ or Cd²⁺ of indicated concentrations. These ions travel through solvent channels within the crystals and chelate with amino acid residues in the selectivity filter region.

X-ray diffraction data were integrated and scaled with the HKL2000 packaged and further processed with the CCP4 package. THe structure of Ca_vAb and its derivatives were solved by molecular replacement by using an individual subunit of the Na_vAb structure (PDB code 3RVY) as the search template. This choice is natural since the proteins used in this study are derived from Na_vAb and differ from it by no more than 3 amino acid residues.

The divalent cations were identified by anomalous difference Fourier maps calculated using data collected at wavelengths of 1.75 Å for Ca²⁺, Cd²⁺ and Mn²⁺. Anomalous scattering describes cases where a pair of structure factors \(\mathbf{F}_{hkl}\) and \(\mathbf{F}_{\overline{hkl}}\) does not obey Friedel’s law², which occurs when the incident X-ray photons have an energy close to a transition energy of the diffracting atom, resulting in absorption of radiation energy and change in phase (normal scattering does not change the phase). At the wavelengths convinient for diffraction, only atoms heavier than phosphorus or sulfur behave as anomalous scatterers. While anomalous scattering data are frequently used to solve the phase problem (in techniques known as single/multiple wavelength anomalous dispersion (SAD/MAD)), it can also be used (as in this study) to calculate an anomalous difference map after phases are available to show the locations of the heavy atoms.³

Crystallography and NMR System software was used for refinement of coordinates and B-factors. Final models were obtained after several cycle refinement with REFMAC and PHENIX and manual re-building using COOT. The geometries of the final structural models of Ca_vAb and its derivatives were verified using PROCHECK.

13 crystal structures of 5 proteins (Na_vAb and 4 Ca_vAb variants) with the following divalent cation concentrations were determined and deposited into PDB:⁴

• TLESWSM (Na_vAb) + Ca²⁺ 15mM
• (TLEDWSM, TLEDESD, TLDDWSM) + Ca²⁺ 15mM
• TLDDWSD + (Ca²⁺ 15mM; Mn²⁺ 100mM; Cd²⁺ 100mM)
• TLDDWSN + Ca²⁺ (0.5, 2.5, 5, 10, 15mM)

Of the 13 crystal structure models, 4MVR (¹⁷⁵TLDDWSD¹⁸¹) corresponds to the protein with the greatest permeability ratio P_Ca:P_Na , while 4MS2 (¹⁷⁵TLDDWSN¹⁸¹) diffracted to the highest resolution (2.75 Å).

Analyses of Results

Ca²⁺ Binding Sites

Using the anomalous diffraction data, the F⁺_Ca - F^-_Ca anomalous difference map was calculated. Two strong peaks followed by a weaker peak were found along the ion-conduction pathway, which correspond to the three Ca²⁺ binding sites. They are designated site 1, 2 and 3 from the extracellular side to the intracellular side.

Site 2 is the site with the highest affinity for Ca²⁺. It is surrounded by a total of 8 oxygen atoms, 4 of which coming from the carboxylate of D177 above and the other 4 from the carbonyl of L176 below. Site 1 is coordinated by the plane of 4 carboxyl groups from D178, and site 3 by the plane of 4 carbonyls from T175 (Figure 2). Throughout the selectivity filter, the O-Ca²⁺ coordination distances are in the range of 4.0-5.0 Å, which is much longer than the ionic diameter of Ca²⁺ (2.28 Å), suggesting that the bound Ca²⁺ ion maintains its hydration shell while passing through the pore. Site 3 has the lowest affinity, consistent with its role in exit of Ca²⁺ from the selectivity filter into the central cavity. Figure 3 shows the molecular model and electron density near the selectivity fileter region of Ca_vAb (TLDDWSN + 15mM Ca²⁺) in COOT⁵ (contoured at r.m.s.d = \(3.02 \sigma\)).

Figure 2: A schemetic showing the Ca²⁺ ion coordination sites of Ca_vAb (TLDDWSD)

Figure 3: The selectivity filter region of 4MS2.

The relative affinities of the three sites were further confirmed by experiments on Ca_vAb (TLDDWSN) with varying Ca²⁺ concentrations (0.5, 2.5, 5, 10, 15mM): at low Ca²⁺ concentration, two strong peaks of approximately equal intensity are found at Site 1 and Site 2; at high concentration the electron density is significantly enhanced in Site 2 and decreased in Site 1. The electron density at Site 3 remains low in all concentrations.