Roles of the lateral fenestration residues of the P2X4 receptor that contribute to the channel function and the deactivation effect of ivermectin

Abstract

P2X receptors are cation-permeable ion channels gated by extracellular adenosine triphosphate (ATP). Available crystallographic data suggest that ATP-binding ectodomain is connected to the transmembrane pore domain by three structurally conserved linker regions, which additionally frame the lateral fenestrations through which permeating ions enter the channel pore. The role of these linker regions in relaying the conformational change evoked by ATP binding of the ectodomain to the pore-forming transmembrane domain has not been investigated systematically. Using P2X 4 R as our model, we employed alanine and serine replacement mutagenesis to determine how the side chain structure of these linker regions influences gating. The mutants Y54A/S, F198A/S, and W259A/S all trafficked normally to the plasma membrane of transfected HEK293 cells but were poorly responsive to ATP. Nevertheless, the function of the F198A/S mutants could be recovered by pretreatment with the known positive allosteric modulator of P2X 4 R, ivermectin (IVM), although the IVM sensitivity of this mutant was significantly impaired relative to wild type. The functional mutants Y195A/S, F200A/S, and F330A/S exhibited ATP sensitivities identical to wild type, consistent with these side chains playing no role in ATP binding. However, Y195A/S, F200A/S, and F330A/S all displayed markedly changed sensitivity to the specific effects of IVM on current deactivation, suggesting that these positions influence allosteric modulation of gating. Taken together, our data indicate that conserved amino acids within the regions linking the ectodomain with the pore-forming transmembrane domain meaningfully contribute to signal transduction and channel gating in P2X receptors.

Molecular structure and function of P2X receptors

“…22 fenestrations (Figure 3C), whereas the effect was increased by the mutation F330A/S, another amino acid of the region (Gao et al, 2015).…”

“…IVM binding sites are believed to be close to the lateral fenestrations found at the membrane interface (Gao et al, 2015;Jiang et al, 2013). It is thus tempting to speculate that IVM acts by insertion between adjacent transmembrane helices, which are located just below these fenestrations, perhaps by slightly moving apart adjacent helices ( Figure 3C).…”

Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

“…Double mutations of P62C/H192C, S65C/S190C, and S65C/D315C (rP2X2 numbering) that restrain the expansion of the lower body markedly attenuated ATP-induced maximal currents of mutant receptors, which was rescued by DTT application [76] . Recently, the linker region between the lower body domain and TMs had also been systematically investigated [77][78][79] , mutations of Y54A, Q55A, F198A, W259A, F324A, and G325A (rP2X4 numbering) resulted in a loss of channel function, suggesting that these residues contribute to the conformational transition from the lower body domain to the TM region. These findings confirm the essential role of the lower body domain in conformational transitions during channel gating.…”

Ivermectin and its target molecules: shared and unique modulation mechanisms of ion channels and receptors by ivermectin

“…IVM alone does not directly activate the P2X 4 receptor but potentiates the amplitude of the ATP‐induced current with an EC 50 of approximately 0.25 μ m (Priel & Silberberg, ; Gao et al . ). IVM also potentiates the ATP‐induced current of the human P2X 7 receptor, but has only a limited effect on the mouse and rat P2X 7 receptors (Norenberg et al .…”

“…; Gao et al . ). Several aromatic residues (Tyr195, Phe198, Phe200 and Phe330) located in the lateral fenestration contribute to IVM sensitivity and thus modulate current deactivation (Gao et al .…”