Plasma membrane cholesterol as a regulator of human and rodent P2X7 receptor activation and sensitization.
Bottom Line: This contrasts with the inhibitory effect of methyl-β-cyclodextrin reported for other P2X subtypes.Mutational analysis suggests the involvement of an N-terminal region and a proximal C-terminal region that comprises multiple cholesterol recognition amino acid consensus (CRAC) motifs, in the cholesterol sensitivity of channel gating.These results reveal cholesterol as a negative regulator of P2X7 receptor pore formation, protecting cells from P2X7-mediated cell death.
Affiliation: From the Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.Show MeSH
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Mentions: Whole-cell currents mediated by the mouse and human P2X7 receptors differ in the degree and rate at which they sensitize in response to exposure to agonist, with human P2X7 showing a more pronounced and slower rate of sensitization (13). We tested the effects of depleting or loading cholesterol on currents activated by repetitive 5-s applications of 300 μm BzATP at both receptors. The mouse P2X7 receptor gave reproducible responses that were strongly inhibited by pretreatment with MCD loaded with cholesterol (Fig. 3, A and B). Treatment with MCD alone dramatically enhanced the amplitude of the current evoked by the first application of agonist and, although subsequent responses were also of larger amplitude, they showed some desensitization, unlike the untreated cells. For the human P2X7 receptor, preincubation with MCD also caused a large increase in the amplitude of the initial currents (Fig. 3, C and D). Under control conditions, recordings showed considerable variability in the degree of run-up (Fig. 3E). Approximately 40% showed a profound run-up after >10 applications of BzATP; for this group, the mean increase was ∼6.1-fold. Following MCD treatment, initial currents were already a similar magnitude to that seen after run-up of untreated cells, and only two recordings subsequently showed a modest run-up. Thus, for both mouse and human P2X7, the effect of MCD is to sensitize the receptor to the initial application of agonist.
Affiliation: From the Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.