N-ethylmaleimide blocks the modulatory effects of divalent cations and guanine nucleotides on the brain substance P receptor.
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 Na2SO4 or 2.5 mM MnCl2) shows a KD of 5.9 and 5.5 nM and a Bmax of 44.4 and 63.9 fmol/mg protein respectively. In the presence of both, there is a 2-fold increase in the affinity (KD 2.8 nM) and a 25-80% increase in the Bmax (81.6 fmol/mg protein). Addition of 100 microM GTP or Gpp(NH)p in either 125 mM Na2SO4 or 2.5 mM MnCl2 or both decreases the Bmax 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 IC50 of 1.0 mM, demonstrating that SH groups play a critical role in the interaction of the ligand with the receptor. If the SP receptors are protected with 1 microM PHY, NEM irreversibly inhibits the effect of divalent cations and guanine nucleotides. Analysis of [3H]PHY binding in 125 mM Na2SO4, 2.5 mM MnCl2 on membranes that were protected with 1 microM PHY and then preincubated with NEM demonstrates a variable decline in receptor number and a 2-fold decrease in the affinity (KD, 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.
D000818:Animals; D001923:Brain Chemistry / Q000187:drug effects*; D002413:Cations, Divalent / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology*; D006150:Guanine Nucleotides / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010803:Physalaemin / Q000378:metabolism; D051381:Rats; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000378:metabolism*
D002413:Cations, Divalent; D006150:Guanine Nucleotides; D009419:Nerve Tissue Proteins; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter; D010803:Physalaemin; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D005033:Ethylmaleimide