Abstract
Vanilloid receptor subunit 1 (VR1) is a nonselective cation channel that integrates multiple pain-producing stimuli. VR1 channels are blocked with high efficacy by the well established noncompetitive antagonist ruthenium red and exhibit high permeability to divalent cations. The molecular determinants that define these functional properties remain elusive. We have addressed this question and evaluated by site-specific neutralization the contribution on pore properties of acidic residues located in the putative VR1 pore region. Mutant receptors expressed in Xenopus oocytes exhibited capsaicin-operated ionic currents akin to those of wild type channels. Incorporation of glutamine residues at Glu(648) and Glu(651) rendered minor effects on VR1 pore attributes, while Glu(636) slightly modulated pore blockade. In contrast, replacement of Asp(646) by asparagine decreased 10-fold ruthenium red blockade efficacy and reduced 4-fold the relative permeability of the divalent cation Mg(2+) with respect to Na(+) without changing the selectivity of monovalent 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 suggest that Asp(646) is a molecular determinant of VR1 pore properties and imply that this residue may form a ring of negative charges that structures a high affinity binding site for cationic molecules at the extracellular entryway.
Highlights
The molecular mechanism underlying chemical and thermal nociception is starting to be understood, thanks to the cloning of a capsaicin-operated neuronal receptor referred to as the vanilloid receptor subunit 1 (VR1)1 [1]
The molecular determinants that define these functional properties remain elusive. We have addressed this question and evaluated by site-specific neutralization the contribution on pore properties of acidic residues located in the putative Vanilloid receptor subunit 1 (VR1) pore region
Neutralization of Asp646 Reduces Ruthenium Red Sensitivity of VR1 Channels—To study the functional role played by negatively charged residues located within or nearby the proposed P-loop of VR1 channels (Fig. 1), we neutralized these acidic amino acids
Summary
Site-directed Mutagenesis, cRNA Preparation, and Microinjection into Xenopus Oocytes—VR1 is a cDNA clone encoding a functional capsaicin-operated channel from dorsal root ganglion [1] (kindly provided by Dr David Julius). Oocytes were transferred to the recording chamber (V ϭ 0.2 ml) and were perfused (1 ml/min) with the appropriate Ringer’s solution in the absence and/or presence of capsaicin as VR1 agonist. Where IC50 is the inhibition constant and denotes the concentration of blocker to inhibit half of the maximal response (Imax) recorded in its absence, and n is the steepness of the inhibition curve. Leak currents were measured in the absence of agonist in the external bath medium and subtracted from the ionic current recorded in its presence. All Ringer’s solutions contained 10 mM Hepes, pH 7.4, and 3.0 mM KCl. Since Mg2ϩ does not activate the endogenous Ca2ϩ-activated, voltagedependent chloride conductance [24], the contribution of ClϪ permeability to the reversal potentials was considered negligible. The GHK equation modified to include the contribution of the permeability to divalent cations is as follows [21, 22], RT
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