The type A gamma amino butyric acid (GABAA) receptor is an important pharmacological target for general anesthetic drugs. Recent progress suggests highly specific effects of several general anesthetics at the GABAA receptor; the details of this mechanism, however, are unknown. Additionally there are many different forms of the subunits which make up the GABAA receptor. The interaction between GABAA receptor subunit composition and general anesthetic pharmacology remains relatively unexplored, and the significance of this interaction to the function of neurons is unknown. This project tests the hypothesis that the GABAA receptor beta-subunit isoform dictates etomidate modulation of GABA-induced current in neurons. The study consists of a series of deliberate steps beginning with a biophysical characterization of the effects of intravenous general anesthetics at GABAA receptors, followed by an examination of the effects of b-subunit gene-targeting on general anesthetic pharmacology and concluding with the creating of neurons with a reversible externally-inducible alteration in sensitivity to etomidate. Specifically, the recently discovered critical role of the beta-subunit and the specific amino acid at location 270 on the beta-subunit will be explored through expression, in HEK293 cells, of different beta-subunit isoforms and point-mutants in an a1bxg2 heteromeric combination. Whole cell patch clamp and rapid GABA perfusion will be used to arrive at a kinetic model of drug action. Next this mechanistic model will be used as a tool to investigate the effects of beta-subunit gene-targeting on general anesthetic pharmacology in retinoic-acid-induced P19 neurons. The P19 system offers an unprecedented opportunity to investigate the effect of gene-targeting without the complexities involved in creating a whole animal. Conventional b1-transgenic, b1-knockout, and conditional gene-targeted neurons will be examined. In the next phase of the research project, the best gene-targeting strategy found in these studies will be used to create a gene-targeted mouse with a reversible externally-inducible alteration in sensitivity to general anesthetics. Detailed understanding of how existing general anesthetics work is essential for the development of improved anesthetics without the very significant side effects of existing agents. The experiments proposed will add quantitative and mechanistic information to general anesthetic molecular pharmacology and move this field of investigation closer towards truly understanding how these clinically essential drugs work.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM052325-09
Application #
6642667
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Cole, Alison E
Project Start
1995-04-01
Project End
2004-06-30
Budget Start
2002-07-03
Budget End
2004-06-30
Support Year
9
Fiscal Year
2002
Total Cost
$277,950
Indirect Cost
Name
Columbia University (N.Y.)
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
167204994
City
New York
State
NY
Country
United States
Zip Code
10032
Yang, Jay; Cheng, Qing; Takahashi, Ayako et al. (2006) Kinetic properties of GABA rho1 homomeric receptors expressed in HEK293 cells. Biophys J 91:2155-62
Hu, Zhihong; Sayeed, Mohammed M (2005) Activation of PI3-kinase/PKB contributes to delay in neutrophil apoptosis after thermal injury. Am J Physiol Cell Physiol 288:C1171-8
Hu, Zhihong; Sayeed, Mohammed M (2004) Suppression of mitochondria-dependent neutrophil apoptosis with thermal injury. Am J Physiol Cell Physiol 286:C170-8
Wagner 2nd, L E; Gingrich, K J; Kulli, J C et al. (2001) Ketamine blockade of voltage-gated sodium channels: evidence for a shared receptor site with local anesthetics. Anesthesiology 95:1406-13
Cestari, I N; Min, K T; Kulli, J C et al. (2000) Identification of an amino acid defining the distinct properties of murine beta1 and beta3 subunit-containing GABA(A) receptors. J Neurochem 74:827-38
Min, K T; Wu, C L; Yang, J (2000) Nondepolarizing neuromuscular blockers inhibit the serotonin-type 3A receptor expressed in Xenopus oocytes. Anesth Analg 90:476-81
Uchida, I; Li, L; Yang, J (1997) The role of the GABA(A) receptor alpha1 subunit N-terminal extracellular domain in propofol potentiation of chloride current. Neuropharmacology 36:1611-21
Cestari, I N; Uchida, I; Li, L et al. (1996) The agonistic action of pentobarbital on GABAA beta-subunit homomeric receptors. Neuroreport 7:943-7
Uchida, I; Cestari, I N; Yang, J (1996) The differential antagonism by bicuculline and SR95531 of pentobarbitone-induced currents in cultured hippocampal neurons. Eur J Pharmacol 307:89-96