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. 2013 Oct 22;110(43):17332-7.
doi: 10.1073/pnas.1316969110. Epub 2013 Oct 7.

Pore waters regulate ion permeation in a calcium release-activated calcium channel

Hao Dong  1 , Giacomo Fiorin, Vincenzo Carnevale, Werner Treptow, Michael L Klein
Affiliations

Pore waters regulate ion permeation in a calcium release-activated calcium channel

Hao Dong et al. Proc Natl Acad Sci U S A. .

Abstract

The recent crystal structure of Orai, the pore unit of a calcium release-activated calcium (CRAC) channel, is used as the starting point for molecular dynamics and free-energy calculations designed to probe this channel's conduction properties. In free molecular dynamics simulations, cations localize preferentially at the extracellular channel entrance near the ring of Glu residues identified in the crystal structure, whereas anions localize in the basic intracellular half of the pore. To begin to understand ion permeation, the potential of mean force (PMF) was calculated for displacing a single Na(+) ion along the pore of the CRAC channel. The computed PMF indicates that the central hydrophobic region provides the major hindrance for ion diffusion along the permeation pathway, thereby illustrating the nonconducting nature of the crystal structure conformation. Strikingly, further PMF calculations demonstrate that the mutation V174A decreases the free energy barrier for conduction, rendering the channel effectively open. This seemingly dramatic effect of mutating a nonpolar residue for a smaller nonpolar residue in the pore hydrophobic region suggests an important role for the latter in conduction. Indeed, our computations show that even without significant channel-gating motions, a subtle change in the number of pore waters is sufficient to reshape the local electrostatic field and modulate the energetics of conduction, a result that rationalizes recent experimental findings. The present work suggests the activation mechanism for the wild-type CRAC channel is likely regulated by the number of pore waters and hence pore hydration governs the conductance.

Keywords: computer simulation; store-operated calcium entry.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Ion binding and structural integrity of the hexameric CRAC channel. (A) Na+ ions (blue) bind to the Glu ring as well as the D182 and the D184 at the extracellular entrance of the wild-type channel. Cl− ions (green) bind to the basic region at the bottom of the channel. The electrostatic potential profile of the pore shows the distinctive charge distribution of the ion permeation pathway. The direction along the membrane normal was defined as the z axis, and the center of lipid bilayers was defined as the origin. Selected pore-lining residues are rendered in stick mode (acidic residues in red, basic residues in blue, and nonpolar residues in white). Selected transmembrane chains are rendered as gray helices, and pore waters are omitted for clarify. (B) Rmsd of Cα atoms for the wild-type (wt) and mutant protein (mt). (C) Rmsf of Cα atoms for the crystal, wt, and mt protein.
Fig. 2.
Fig. 2.
Shown are the distribution of waters (red), ions (blue and green), and key residues (black and orange), respectively, in the pore of both the wild-type (Upper, left to right) and the mutant (Lower). Note the higher density of water (Left) in the range from 0 Å to ∼10 Å, due to the substitution V174A in the mutant. Overall, the positions of ions and key residues in the two systems are quite similar, indicating relatively small conformational changes after structural relaxation. The histograms were taken from MD trajectories and include all of the species within 10 Å of the pore axis.
Fig. 3.
Fig. 3.
Correlations between the water dipole moment and its position along the membrane normal, Z (Å) in (A) the wild-type (wt) and (B) the mutant (mt) channels. The orientation of waters is asymmetric in the hydrophobic section. The concerted water dipole is stronger in the wt than in the mt, suggesting that water in the mt has freedom to reorient, thus facilitating the transport of ions through the channel. The histograms were taken from MD trajectories and include all of the waters within 10 Å of the pore axis. (C) Averaged water dipole along the membrane normal. (D) Averaged number of waters in the pore. (E) Projected water dipole along the pore axis.
Fig. 4.
Fig. 4.
Shown are free-energy profiles for Na+ permeation through both the wild-type channel (red) and the mutant (blue). The free energy calculated without and with the external field corrections are drawn in solid and dashed lines, respectively. In both cases, the mutant has a more favorable permeation pathway than the wild type. The free-energy profile of the mutant indicates a relatively broad gating region, ranging from A174 to R155. (Inset) Membrane potential of the two systems. The passage of Na+ in the mutant was found to experience a lower barrier due to the intrinsic field from the protein, which is consistent with the free-energy profiles. Two representative snapshots show the major well (Lower Left) and barrier (Lower Right) experienced by the Na+ ion along the permeation pathway in the mutant. Color coding is the same as in Fig. 1, except for the Na+ driven to move, which is shown in magenta. For visual clarity, only four of the six TM1 helices are shown.
See this image and copyright information in PMC

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