Identification of an aspartic residue in the P-loop of the vanilloid receptor that modulates pore properties

Vanilloid receptor subunit 1 (VR1) is a nonselective cation channel that in tegrates multiple pain-producing stimuli. VR1 channels are blocked with hig h efficacy by the well established noncompetitive antagonist ruthenium red and exhibit high permeability to divalent cations. The molecular determinan ts that define these functional properties remain elusive. We have addresse d this question and evaluated by site-specific neutralization the contribut ion on pore properties of acidic residues located in the putative VR1 pore region; Mutant receptors expressed in Xenopus oocytes exhibited capsaicin-o perated ionic currents akin to those of wild type channels. Incorporation o f glutamine residues at Glu(648) and Glu(651) rendered minor effects on VR1 pore attributes, while Glu(636) slightly modulated pore blockade. In contr ast, replacement of Asp(646) by asparagine decreased 10-fold ruthenium red blockade efficacy and reduced 1-fold the relative permeability of the dival ent cation Mg2+ with respect to Na+ without changing the selectivity of mon ovalent cations, At variance with wild type channels and E636Q, E648Q, and E651Q mutant receptors, ruthenium red blockade of D646N mutants was weakly sensitive to extracellular pH acidification. Collectively, our results sugg est that Asp(646) is a molecular determinant of VR1 pore properties and imp ly that this residue may form a ring of negative charges that structures a high affinity binding site for cationic molecules at the extracellular entr yway.