Increased Hydrophobicity at the N Terminus/Membrane Interface Impairs Gating of the Severe Combined Immunodeficiency-related ORAI1 Mutant

Abstract

Patients with severe combined immune deficiency (SCID) suffer from defective T-cell Ca 2؉ signaling. A loss of Ca 2؉ entry has been linked at the molecular level to single missense mutation R91W in the store-operated Ca 2؉ channel ORAI1. However, the mechanistic impact of this mutation on ORAI1 function remains unclear. Confocal Förster resonance energy transfer microscopy revealed that dynamic store-operated coupling of STIM1 to ORAI1 R91W was largely sustained similar to wild-type ORAI1. Characterization of various point mutants at position 91 by whole cell patch clamp recordings displayed that neutral or even negatively charged amino acids did not abolish ORAI1 function. However, substitution by hydrophobic leucine, valine, or phenylalanine resulted in non-functional ORAI1 channels, despite preserved STIM1 coupling. Besides conformational constraints at the N terminus/membrane interface predicted for the hydrophobic mutants, additional key factor(s) were suggested to determine ORAI1 functionality. Calculation of the probability for the 1st transmembrane domain and its hydrophobicity revealed a substantial increase for all hydrophobic substitutions that lead to non-functional ORAI1 R91X mutants in contrast to those with hydrophilic residues. Hence, increased hydrophobicity might lead to disrupted permeation/gating, as an ORAI1 channel with increased pore size and R91W mutation failed to recover activity. In conclusion, the increase in hydrophobicity at the N terminus/ membrane interface represents the major cause for yielding nonfunctional ORAI1 channels.The immune system consists of various cell types such as Tand B-cells that are involved in protecting the body from foreign particles and pathogenic organisms. Defects in T-cell development impair normal immune function and may lead to primary immune deficiency. One subgroup thereof represented by the severe combined immunodeficiency (SCID) 4 occurs in 1 of 50,000 -100,000 live births, causing an onset of one or more serious infections, such as pneumonia, meningitis, or bloodstream infections, within the first few months of life (1, 2). It is currently known that defective T-cell signaling in SCID patients can arise from mutations in different genes including a point mutation in ORAI1 (3-5). T-cell function and proliferation requires calcium influx mediated by the Ca 2ϩ release-activated Ca 2ϩ channel. It is activated by depletion of intracellular Ca 2ϩ stores induced by the second messenger inositol 1,4,5-trisphosphate (6 -9) and this cytosolic Ca 2ϩ entry serves essential functions from secretion to gene expression and cell growth (10).A combination of RNA interference-based screening and analysis of single nucleotide polymorphism arrays of patients with SCID syndrome has led to the identification of the plasmamembrane protein ORAI1 as a key component of the Ca 2ϩ release-activated Ca 2ϩ channel complex (11,12). An overexpression of wild-type ORAI1 (4) or a related member ORAI3 (13) in SCID T-cells partially restored store-operated Ca 2ϩ influx. Based on p...

Pore architecture of the ORAI1 store-operated calcium channel

“…R91 itself is not essential, as evidenced by the normal currents carried by R91E and R91G channels (35), and therefore the defect in the R91W channel has been interpreted as interference by the bulky tryptophan side chain with channel gating or with ion flux through the pore (35)(36)(37)(38). Other substitutions of interest are R91T, which reduces the amplitude of whole-cell currents compared to wild-type ORAI1; and S89G/S90G, which increases currents (35). In the two latter cases, the inward rectifying current-voltage (I-V) curve in standard recording conditions implies that the substitutions do not alter the selectivity filter (35).…”

“…Attention has focused mainly on ORAI1 channels with the SCID mutation R91W (2) or with the experimental replacements R91V, R91L, or R91F (35), which fail to carry current upon store depletion even though the proteins are at the cell surface and interact with STIM1 (35)(36)(37)(38). R91 itself is not essential, as evidenced by the normal currents carried by R91E and R91G channels (35), and therefore the defect in the R91W channel has been interpreted as interference by the bulky tryptophan side chain with channel gating or with ion flux through the pore (35)(36)(37)(38). Other substitutions of interest are R91T, which reduces the amplitude of whole-cell currents compared to wild-type ORAI1; and S89G/S90G, which increases currents (35).…”

The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca2+-dependent inactivation

“…As an alternative gating mechanism a ring of W76 side chains could be brought together during CDI to function as a hydrophobic gate, by analogy to the block of conduction by the Orai1 R91W pore mutation (Feske et al, 2006;Derler et al, 2009a). W76 could also stabilize pore conformations in which other residues form the gate, such as V102 and the adjacent hydrophobic region (McNally et al, 2012;Gudlur et al, 2014).…”

Store-Operated Calcium Channels: New Perspectives on Mechanism and Function

“…Interestingly, K85E does not prevent CAD binding to the amino terminus, suggesting that this residue is required for allosteric changes leading to gate opening. R91 may serve a similar function (Derler et al 2009a). Interestingly, this membraneproximal amino-terminal region overlaps with a calmodulin (CaM) binding site (aa 68 -91), and several residues in this region (A73, Y80, W76) appear to be essential for CaM binding and Ca 2þ -dependent inactivation (Fig.…”

“…Deletion of aa 73 -84 prevents CAD binding to the amino terminus and channel opening ). Mutations such as K85E (Lis et al 2010) and R91W, the mutation found originally in T cells from SCID patients , completely prevent channel opening, apparently without preventing STIM1-Orai1 binding (Navarro-Borelly et al 2008;Derler et al 2009a), although R91W may interact with STIM1 somewhat less strongly than wild-type Orai1 (Fig. 3) (Muik et al 2011).…”