Multiple determinants of dihydro-beta-erythroidine sensitivity on rat neuronal nicotinic receptor alpha subunits.
Neuronal nicotinic acetylcholine receptors are differentially sensitive to blockade by the competitive antagonist dihydro-beta-erythroidine. Both alpha and beta subunits participate in determining sensitivity to this antagonist. The alpha subunit contribution to dihydro-beta-erythroidine sensitivity is illustrated by comparing the alpha 4 beta 4 receptor and the alpha 3 beta 4 receptor, which differ in sensitivity to dihydro-beta-erythroidine by approximately 120-fold. IC50 values for blocking alpha 4 beta 4 and alpha 3 beta 4, responding to EC20 concentrations of acetylcholine, were 0.19 +/- 0.06 and 23.1 +/- 10.2 microM, respectively. To map the sequence segments responsible for this difference, we constructed a series of chimeric alpha subunits containing portions of the alpha 4 and alpha 3 subunits. These chimeras were coexpressed with beta 4, allowing pharmacological characterization. We found determinants of dihydro-beta-erythroidine sensitivity to be distributed throughout the N-terminal extracellular domain of the alpha subunit. These determinants were localized to sequence segments 1-94, 94-152, and 195-215. Loss of determinants within segment 1-94 had the largest effect, decreasing dihydro-beta-erythroidine sensitivity by 4.3-fold.
Harvey
S C
SC
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine 33101, USA.
Maddox
F N
FN
Luetje
C W
CW
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D004079:Dihydro-beta-Erythroidine / Q000494:pharmacology*; D005260:Female; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects; D009474:Neurons / Q000502:physiology*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D014982:Xenopus laevis
D046911:Macromolecular Substances; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D004079:Dihydro-beta-Erythroidine; D000109:Acetylcholine