Pharmacology and Neuroscience

Dr. Michael P. Blanton

Professor of Pharmacology
Ph.D., 1989, University of California, Santa Cruz

Structure/Function of Ligand-Gated Ion Channels and P-type Ion-Motive ATPases:

LGIC: The nicotinic acetylcholine, serotonin (5-HT₃), γ-aminobutyric acid type A (GABA_A), and glycine receptors belong to a superfamily of ligand-gated ion channels (LGIC) each being critical for rapid signal transduction in the nervous system. In addition, each of these receptors constitute important target sites for many therapeutic drugs. Research in the lab is focused on two overall objectives: First, to determine how each of these ligand-gated ion channels function. More specifically, to identify and characterize the different structural elements, including the lipid-protein interface, that mediate each aspect of receptor function. To determine how each of these elements interact to effect the overall functioning of the receptor. Inclusive in this first goal is work aimed at determining in detail the structure of AChR functional elements (e.g. ion channel, lipid-protein interface. Secondly, to determine how drugs, in particular both local and general anesthetics (and neurosteroids), interact with each of the LGIC family members. Inclusive in this goal is not only understanding what are the determinants of drug binding but what are the mechanisms by which the binding of these ligands effect the structure of the receptor both locally as well as distally (i.e. allosterically). To accomplish these goals a wide array of techniques are employed in the laboratory and with collaborators, including: photoaffinity labeling, protein chemistry, spectroscopy (CD, FTIR, Fluorescence), as well as molecular biological and electrophysiological techniques.

Figure 1: Molecular Model of the barbiturate Pentobarbital complexed with the Nicotinic Acetylcholine Receptor Ion Channel. (from Arias et al., (2001) Molecular Pharmacology 60, 497-506)

Na,K-ATPases: The Na,K-ATPase is present in the plasma membrane of nearly all animal cells where it couples the hydrolysis of ATP to the transport of Na⁺ ions out of cells and K⁺ ions into cells, thereby establishing an electrochemical gradient. The ion gradients created by the Na,K-ATPase are fundamental to many diverse cellular functions, including fluid and electrolyte balance in animals. Research in the lab is focused on two major goals: 1) Identify membrane-spanning segments and individual amino acid residues which contribute to the formation of the lipid-protein interface of the ATPase. The research design is to covalently tag lipid-exposed residues of the Na,K-ATPase using the hydrophobic photoreactive compound 3-trifluoromethyl-3-(m-[¹²⁵I]iodophenyl) diazirine ([¹²⁵I]TID) as well as with photoreactive lipid analogs of cholesterol and phosphatidylserine. Lipid-exposed segments and amino acids are to be determined by N-terminal sequencing of isolated proteolytic fragments. Furthermore, preliminary results with [¹²⁵I]TID have established that the extent of labeling of the Na,K-ATPase is conformationally-sensitive. There is increased [¹²⁵I]TID incorporation into the Na,K-ATPase in the E₂ conformation and the enhanced labeling maps to proteolytic fragments which contain the M5 and M6 transmembrane segments. 2) The second goal is to then identify [¹²⁵I]TID labeled residues in the M5 and M6 segments in both the E₁ and E₂ conformations of each ATPase, residues which are putatively located in the cation occlusion domain of each ATPase and are therefore part of the cation translocation pathway.

Figure 2: Mapping the sites of [125I]TID photoincorporation into the Na,K-ATPase (from Blanton and McCardy (2000) Biochemistry 39, 13534-13544).

Representative Publications

(Recent publications) Click on the pdf file to view the publications.

Mitesh Sanghvi, Ayman K. Hamouda, Shouryadeep Srivastava, Margaret I. Davis, David C. Chiara, Tina K. Machu, David M. Lovinger, Jonathan B. Cohen, and Michael P. Blanton (2009)
Hydrophobic Photolabeling Studies Identify the Lipid-Protein Interface of the 5-HT_3AR. Biochemistry 48, 9278-9286.

Shouryadeep Srivastava, Ayman K. Hamouda, Akash Pandhare, Phaneendra K. Duddempudi, Mitesh Sanghvi, Jonathan B. Cohen, and Michael P. Blanton. (2009)
[³H]Epibatidine Photolabels Non-Equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors. Journal of Biological Chemistry (JBC) 284, 24939-24947.

Ayman K. Hamouda, Xiaochun Jin, Mitesh Sanghvi, Shouryadeep Srivastava, Akash Pandhare, Phaneendra K. Duddempudi, Joe Henry Steinbach, and Michael P. Blanton. (2009)
Photoaffinity Labeling the Agonist Binding Domain of α4β4 and α4β2 Neuronal Nicotinic Acetylcholine Receptors with [¹²⁵I]Epibatidine and 5[¹²⁵I]A-85380. Biochim. Biophys Acta, 1788, 1987-1995.

Ayman K. Hamouda, Mitesh Sanghvi, David C. Chiara, Jonathan B. Cohen, and Michael P. Blanton (2007)
Identifying the Lipid-Protein Interface of the α4β2 Neuronal Nicotinic Acetylcholine Receptor: Hydrophobic Photolabeling Studies with 3-(Trifluoromethyl)-3-(m-[¹²⁵I]iodophenyl)diazirine. Biochemistry 46: 13837-13846.

Ayman Hamouda, David Chiara, Daniel Sauls, Jonathan B. Cohen, and Michael P. Blanton (2006) Cholesterol Interacts with Transmembrane α-helices M1, M3, and M4 of the Torpedo Nicotinic Acetylcholine Receptor: Photolabeling Studies using [3H]Azicholesterol. Biochemistry 45: 976-986.

Ayman Hamouda, Mitesh Sangvhi, Nelli Vardanyan, Tina Machu, and Michael P. Blanton (2006) Assessing the Lipid Requirements of the Torpedo californica Nicotinic Acetylcholine Receptor. Biochemistry 45: 4327-4337.

John F. Leite, Michael P. Blanton, Mona Shahgholi, Dennis Doughery, and Henry A. Lester. (2003) Conformational State-Dependent Hydrophobic Photolabeling of the Nicotinic Acetylcholine Receptor Using Electrophysiology-Coordinated Photochemistry and Mass Spectrometry. PNAS 100, 13054-13059.

Hugo R. Arias, James R. Trudell, Erin Z. Bayer, Brent Hester, Elizabeth McCardy, and Michael P. Blanton. (2003) Noncompetitive Antagonist Binding Sites in the Torpedo Nicotinic Acetylcholine Receptor Ion Channel Structure-Activity Relationship Studies Using Adamantane Derivatives. Biochemistry 42, 7358-7370.

DaCosta, C.J.B., Ogrel, A.A., McCardy, E.A., Blanton, M.P., Baenziger, J.E. (2002) Lipid-Protein Interactions at the Nicotinic Acetylcholine Receptor: A Functional Coupling Between Nicotinic Receptors and Phosphatidic Acid-Containing Lipid Bilayers. J. Biol. Chem. 277, 201-208.

Hugo R. Arias, Elizabeth A. McCardy, Martin J. Gallagher, and Michael P. Blanton. (2001) Interaction of Barbiturate Analogs with the Nicotinic Acetylcholine Receptor Channel. Molecular Pharmacology 60, 497-506.

Natalie Methot, Blair D. Ritchie, Michael P. Blanton and John E. Baenziger (2001) Structure of the Pore-forming Transmembrane Domain of a Ligand-gated Ion Channel. J Biol Chem, 276, 23726-23732.

Michael P. Blanton, S.K. Raja, Anil K. Lala, and Jonathan B. Cohen.(2001) Identification and Characterization of Membrane-associated Polypeptides in Torpedo Nicotinic Acetylcholine Receptor-rich Membranes by Hydrophobic Photolabeling BBA, Biomembranes 1512(2), 215-224.

Hugo R. Arias, Elizabeth A. McCardy, and Michael P. Blanton. (2001) Characterization of the Dizoclipine Binding Site on the Nicotinic Acetylcholine Receptor.Molecular Pharmacology 59, 1051-1060.

Stephen E. Ryan, Michael P. Blanton and John E. Baenziger.(2001) A Conformational Intermediate between the Resting and Desensitized States of the Nicotinic Acetylcholine Receptor. J. Biol. Chem. 276, 4796-4803.

Francisco J. Barrantes, Silivia S. Antollini, Michael P. Blanton, and Manuel Prieto. (2000) Topography of the nicotinic acetylcholine receptor membrane-embedded domains. J. Biol. Chem. 275, 37414-37422.

Michael P. Blanton and Elizabeth A. McCardy. (2000) Identifying the Lipid-Protein Interface and Transmembrane Structural Transitions of the Torpedo Na,K-ATPase using Hydrophobic Photoreactive Probes. Biochemistry 39, 13534-13544.

Hugo R. Arias and Michael P. Blanton. a-Conotoxins.(2000) The International Journal of Biochemistry and Cell Biology 32(10), 1017-1028.

Michael P. Blanton, Elizabeth A. McCardy, and Martin J. Gallagher. (2000) Examining the Noncompetitive Antagonist Binding Site in the Ion Channel of the Nicotinic Acetylcholine Receptor in the Resting State. J. Biol. Chem. 275, 3469-3478.

Michael P. Blanton, Elizabeth McCardy, Minghua Liu, John D. Fryer, and Ronald J. Lukas. (2000) 5-Hydroxytryptamine Interaction with the Nicotinic Acetylcholine Receptor. Eur. J. Pharmacol. 389 (2-3), 155-163.

Michael P. Blanton, Yu Xie, Larry Dangott, and Jonathan B. Cohen. (1999) The Steroid Promegestone is a Noncompetitive Antagonist of Torpedo Nicotinic Acetylcholine Receptor which Interacts with the Lipid:Protein Interface. Mol. Pharm. 55 , 269-278.

John Corbin, Nathalie Methot, Howard H. Wang, John E. Baenziger, & Michael P. Blanton. (1998) Secondary Structure Analysis of Individual Transmembrane Segments of the Nicotinic Acetylcholine Receptor by CD and FTIR Spectroscopy. J. Biol. Chem. 273, 771-777.

Michael P. Blanton, D. Parikh, A. Huggins, and E.A. McCardy. (1998) Probing the Structure of the Nicotinic Acetylcholine Receptor with the Hydrophobic Photoreactive Probes [125I]TID-BE and [125I]TIDPC/16 Biochemistry 37, 14545-14555.

Michael P. Blanton, Larry Dangott, S.K. Raja ,Anil K. Lala, and Jonathan B. Cohen. (1998) Probing the Structure of the Torpedo Nicotinic Acetylcholine Receptor Ion Channel with the Uncharged Photoactivatable Compound [3H] Diazofluorene. J. Biol. Chem 273, 8659-8668.

John Corbin, Howard H. Wang and Michael P. Blanton. (1998) Identifying the Cholesterol Binding Domain in the Nicotinic Acetylcholine Receptor with [125I]Azido-Cholesterol.(BBA, Biomembranes,1414 (1-2), 65-74.

For further information contact Dr. Michael P. Blanton

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