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

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.store-operated calcium entry | computer simulation C alcium release-activated calcium (CRAC) channels in the plasma membrane are integral membrane proteins that play a central role in cellular signaling by generating the sustained influx of calcium (1-3). Immune response in cells consists typically of a fall in Ca 2+ content within the endoplasmic reticulum (ER), followed by the opening of the store-operated CRAC channels that leads to the sustained increase in intracellular Ca 2+ concentrations (4). Despite about two decades of research, the molecular components underlying this process of store-operated calcium entry (SOCE) have been unknown until recently, when the stromal interaction molecule (STIM) was determined to be the ER Ca 2+ sensor (5, 6) that activates the channel in response to the depletion of intracellular calcium store content. Later, the Orai protein was identified as the pore subunit of the channel (7-9). CRAC regulator 2A was then discovered to reinforce the binding of the two aforementioned key components at elevated Ca 2+ levels to promote the SOCE, and to inhibit the influx at low Ca 2+ levels by dissociating from the complex (10). The newly published crystal structure of the CRAC channel pore subunit, Orai, from Drosophila melanogaster at 3.35 Å resolution, provides groundbreaking insights into a molecular architecture that is distinct from ...

Ab initio theory and modeling of water

“…In conclusion, the SCAN XC functional within DFT shows promising predictive power and will likely enable confident ab initio predictions for complex systems at the forefront of physics, chemistry, biology, and materials science (47).…”

Alternative splicing converts STIM2 from an activator to an inhibitor of store-operated calcium channels

“…In principle, the reduced current could reflect inhibition of channel opening or inhibition of ion flow through the open pore. Interestingly, Orai1(V102C/A) channels are thought to acquire their constitutive activity from the removal of a hydrophobic barrier to ion permeation near V102 (McNally et al, 2012;Dong et al, 2013;Gudlur et al, 2014). Thus, the ability of the tethered STIM2β SS construct to inhibit Ca 2+ flux through Orai1(V102C) supports the latter possibility, that STIM2β inhibits Orai1 by imposing a new barrier to conduction, although additional effects on channel opening cannot be ruled out.…”

The STIM-Orai Pathway: Orai, the Pore-Forming Subunit of the CRAC Channel

“…It is anchored by the region from residues 99–104, whose relative structural rigidity is evidenced by low rotational mobility in the intermonomer disulfide crosslinking assay and by low temperature factors in the corresponding region of the Drosophila Orai crystal structure (Zhou et al 2010b; Hou et al 2012). The nonpolar segment of the pore presents a barrier to ion flux that can be traced in silico in the free energy profile for Na + traversing the closed wildtype channel (Dong et al 2013) and experimentally in the barrier that prevents constitutive Ca 2+ flux in a channel truncated to remove other proposed barriers at R91 and in the TM1 cytoplasmic extensions (Gudlur et al 2014). The several lines of evidence that this barrier is displaced during STIM-dependent channel gating are discussed below.…”

“…Replacement of V102 by the small and more polar residues C, S, T, G, or A results in a constitutively conducting and less selective channel (McNally et al 2012; Derler et al 2013). In silico calculations for the Drosophila V174A channel—corresponding to a human V102A channel—suggest that it retains the closed pore conformation of the wildtype Drosophila channel but presents a markedly lower energetic barrier to ion conductance (Dong et al 2013). Consistent with the idea that the channel is not already in the STIM-gated conformation, the relatively nonselective V102X channels undergo a STIM-dependent conformational change to more selective channels (McNally et al 2012; Derler et al 2013).…”