α-Conotoxins Identify the α3β4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells.
Ligands that selectively inhibit human α3β2 and α6β2 nicotinic acetylcholine receptor (nAChRs) and not the closely related α3β4 and α6β4 subtypes are lacking. Current α-conotoxins (α-Ctxs) that discriminate among these nAChR subtypes in rat fail to discriminate among the human receptor homologs. In this study, we describe the development of α-Ctx LvIA(N9R,V10A) that is 3000-fold more potent on oocyte-expressed human α3β2 than α3β4 and 165-fold more potent on human α6/α3β2β3 than α6/α3β4 nAChRs. This analog was used in conjuction with three other α-Ctx analogs and patch-clamp electrophysiology to characterize the nAChR subtypes expressed by human adrenal chromaffin cells. LvIA(N9R,V10A) showed little effect on the acetylcholine-evoked currents in these cells at concentrations expected to inhibit nAChRs with β2 ligand-binding sites. In contrast, the β4-selective α-Ctx BuIA(T5A,P6O) inhibited >98% of the acetylcholine-evoked current, indicating that most of the heteromeric receptors contained β4 ligand-binding sites. Additional studies using the α6-selective α-Ctx PeIA(A7V,S9H,V10A,N11R,E14A) indicated that the predominant heteromeric nAChR expressed by human adrenal chromaffin cells is the α3β4* subtype (asterisk indicates the possible presence of additional subunits). This conclusion was supported by polymerase chain reaction experiments of human adrenal medulla gland and of cultured human adrenal chromaffin cells that demonstrated prominent expression of RNAs for α3, α5, α7, β2, and β4 subunits and a low abundance of RNAs for α2, α4, α6, and α10 subunits.
Departamento de Farmacología y Terapéutica, Universidad Autónoma de Madrid, Madrid, Spain (A.J.H., A.A.); Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M., L.A.); George E. Whalen Veterans Affairs Medical Center, Salt Lake City, Utah (J.M.M.); Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania (J.L.); Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (L.L., P.W.); Hospital Doce de Octubre, Madrid, Spain (J.P.); and Hospital Clínico San Carlos Madrid, Spain (J.B.).
Arik J
AJ
Hone
Departamento de Farmacología y Terapéutica, Universidad Autónoma de Madrid, Madrid, Spain (A.J.H., A.A.); Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M., L.A.); George E. Whalen Veterans Affairs Medical Center, Salt Lake City, Utah (J.M.M.); Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania (J.L.); Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (L.L., P.W.); Hospital Doce de Octubre, Madrid, Spain (J.P.); and Hospital Clínico San Carlos Madrid, Spain (J.B.).
J Michael
JM
McIntosh
Departamento de Farmacología y Terapéutica, Universidad Autónoma de Madrid, Madrid, Spain (A.J.H., A.A.); Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M., L.A.); George E. Whalen Veterans Affairs Medical Center, Salt Lake City, Utah (J.M.M.); Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania (J.L.); Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (L.L., P.W.); Hospital Doce de Octubre, Madrid, Spain (J.P.); and Hospital Clínico San Carlos Madrid, Spain (J.B.).
Layla
L
Azam
Departamento de Farmacología y Terapéutica, Universidad Autónoma de Madrid, Madrid, Spain (A.J.H., A.A.); Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M., L.A.); George E. Whalen Veterans Affairs Medical Center, Salt Lake City, Utah (J.M.M.); Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania (J.L.); Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (L.L., P.W.); Hospital Doce de Octubre, Madrid, Spain (J.P.); and Hospital Clínico San Carlos Madrid, Spain (J.B.).
Jon
J
Lindstrom
Departamento de Farmacología y Terapéutica, Universidad Autónoma de Madrid, Madrid, Spain (A.J.H., A.A.); Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M., L.A.); George E. Whalen Veterans Affairs Medical Center, Salt Lake City, Utah (J.M.M.); Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania (J.L.); Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (L.L., P.W.); Hospital Doce de Octubre, Madrid, Spain (J.P.); and Hospital Clínico San Carlos Madrid, Spain (J.B.).
Linda
L
Lucero
Departamento de Farmacología y Terapéutica, Universidad Autónoma de Madrid, Madrid, Spain (A.J.H., A.A.); Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M., L.A.); George E. Whalen Veterans Affairs Medical Center, Salt Lake City, Utah (J.M.M.); Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania (J.L.); Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (L.L., P.W.); Hospital Doce de Octubre, Madrid, Spain (J.P.); and Hospital Clínico San Carlos Madrid, Spain (J.B.).
Paul
P
Whiteaker
Departamento de Farmacología y Terapéutica, Universidad Autónoma de Madrid, Madrid, Spain (A.J.H., A.A.); Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M., L.A.); George E. Whalen Veterans Affairs Medical Center, Salt Lake City, Utah (J.M.M.); Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania (J.L.); Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (L.L., P.W.); Hospital Doce de Octubre, Madrid, Spain (J.P.); and Hospital Clínico San Carlos Madrid, Spain (J.B.).
Juan
J
Passas
Departamento de Farmacología y Terapéutica, Universidad Autónoma de Madrid, Madrid, Spain (A.J.H., A.A.); Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M., L.A.); George E. Whalen Veterans Affairs Medical Center, Salt Lake City, Utah (J.M.M.); Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania (J.L.); Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (L.L., P.W.); Hospital Doce de Octubre, Madrid, Spain (J.P.); and Hospital Clínico San Carlos Madrid, Spain (J.B.).
Jesús
J
Blázquez
Departamento de Farmacología y Terapéutica, Universidad Autónoma de Madrid, Madrid, Spain (A.J.H., A.A.); Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M., L.A.); George E. Whalen Veterans Affairs Medical Center, Salt Lake City, Utah (J.M.M.); Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania (J.L.); Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (L.L., P.W.); Hospital Doce de Octubre, Madrid, Spain (J.P.); and Hospital Clínico San Carlos Madrid, Spain (J.B.) almudena.albillos@uam.es.
Almudena
A
Albillos
D000313:Adrenal Medulla / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism*; D020916:Conotoxins / Q000494:pharmacology*; D006801:Humans; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D020033:Protein Isoforms; D051381:Rats; D011978:Receptors, Nicotinic / Q000145:classification / Q000378:metabolism*; D014982:Xenopus laevis
D020916:Conotoxins; D018733:Nicotinic Antagonists; D020033:Protein Isoforms; D011978:Receptors, Nicotinic; C403530:nicotinic receptor alpha3beta4