N-ethylmaleimide blocks the modulatory effects of divalent cations and guanine nucleotides on the brain substance P receptor

Abstract

The binding of [ 3H]physalaemin ([ 3H]PHY) to rat brain substance P receptors is modulated by cations and guanine nucleotides. [ 3H]PHY binding in the presence of either monovalent or divalent cations (125 mM Na 2SO 4 or 2.5 mM MnCl 2) shows a K D of 5.9 and 5.5 nM and a B max of 44.4 and 63.9 fmol/mg protein respectively. In the presence of both, there is a 2-fold increase in the affinity (K D 2.8 nM) and a 25–80% increase in the B max (81.6 fmol/mg protein). Addition of 100 μM GTP or Gpp(NH)p in either 125 mM Na 2SO 4 or 2.5 mM MnCl 2 or both decreases the B max by 25–55%. However, the receptor affinity for [ 3H]PHY is not significantly altered by guanine nucleotides. N-Ethylmaleimide (NEM) irreversibly inhibits the receptor binding with an IC 50 of 1.0 mM, demosntrating that SH groups play a critical role in the interaction of the ligand with the receptor. If the SP receptors are protected with 1 μM PHY, NEM irreversibly inhibits the effect of divalent cations and guanine nucleotides. Analysis of [ 3H]PHY binding in 125 mM Na 2SO 4, 2.5 mM MnCl 2 on membranes that were protected with 1 μM PHY and then preincubated with NEM demonstrates a variable decline in receptor number and a 2-fold decrease in the affinity (K D, from 2.8 to 6.9 nM). These observations indicate the existence of a second class of SH groups that are essential for the interaction of divalent cations and guanine nucleotides with the receptor. The blockade of the modulatory effects of divalent cations and guanine nucleotides by NEM treatment further suggests that brain SP receptors are coupled to a guanine nucleotide binding regulatory protein.