Voltage-gated Ca2+-channels (Cav) and protein kinase C (PKC) can both be regulated by volatile anesthetics, but the nature of the effect has been controversial. We hypothesize that anesthetic inhibition of Cav channels and synaptic transmission are mediated by specific PKC isozymes. Although potential PKC phosphorylation sites are present in the pore-forming alpha1 and the auxiliary beta and alpha2/delta subunits of Cav channels; alpha1subunits seem to play the major role in PKC-induced regulation of these channels.
In Aim I of this proposal, we will identify phosphorylation sites in the a1 subunits of Cav channels by direct (with phorbol 12-myristate 13-acetate; PMA) or receptor-stimulated (with muscarinic M1) activation of PKC in Xenopus oocytes coexpressing these receptors and Cav channels. Possible PKC phosphorylation sites will be mutated to Ala or Glu and whole-cell Ba2+ (a substitute for Ca2+)-current in response to two types of PKC activation will be investigated using voltage-clamp measurements.
In Aim II we will identify PKC isozymes involved in the direct or receptor-induced modulation of PKC-sensitive Cav channels expressed in Xenopus oocytes. This will be examined using experiments involving down-regulation of PKC, activation-induced translocation, selective loss/inhibition of individual PKC isozymes and 'add-back' studies.
In Aim III we will identify the PKC isozymes modulated by volatile anesthetics, halothane or isoflurane in Xenopus oocytes by employing the methods mentioned in Aim II. Based on the results of our studies, isozyme selective activators or inhibitors of PKC may be used along with the volatile general anesthetics to i) potentiate the action of anesthetics and ii) to reduce the side effects of these agents caused by their non-specific actions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065214-03
Application #
6927927
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Cole, Alison E
Project Start
2003-08-01
Project End
2008-07-31
Budget Start
2005-08-01
Budget End
2006-07-31
Support Year
3
Fiscal Year
2005
Total Cost
$215,679
Indirect Cost
Name
University of Virginia
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Rajagopal, S; Fang, H; Lynch 3rd, C et al. (2011) Effects of isoflurane on the expressed Cav2.2 currents in Xenopus oocytes depend on the activation of protein kinase C? and its phosphorylation sites in the Cav2.2?1 subunits. Neuroscience 182:232-40
Rajagopal, Senthilkumar; Fang, Hongyu; Lynch 3rd, Carl et al. (2010) Formalin-induced short- and long-term modulation of Cav currents expressed in Xenopus oocytes: an in vitro cellular model for formalin-induced pain. Basic Clin Pharmacol Toxicol 106:338-47
Rajagopal, S; Fang, H; Oronce, C I A et al. (2009) Site-specific regulation of CA(V)2.2 channels by protein kinase C isozymes betaII and epsilon. Neuroscience 159:618-28
Rajagopal, Senthilkumar; Fang, Hongyu; Patanavanich, Saharat et al. (2008) Protein kinase C isozyme-specific potentiation of expressed Ca v 2.3 currents by acetyl-beta-methylcholine and phorbol-12-myristate, 13-acetate. Brain Res 1210:1-10
Fang, Hongyu; Patanavanich, Saharat; Rajagopal, Senthilkumar et al. (2006) Inhibitory role of Ser-425 of the alpha1 2.2 subunit in the enhancement of Cav 2.2 currents by phorbol-12-myristate, 13-acetate. J Biol Chem 281:20011-7
Fang, Hongyu; Franke, Ruthie; Patanavanich, Saharat et al. (2005) Role of alpha1 2.3 subunit I-II linker sites in the enhancement of Ca(v) 2.3 current by phorbol 12-myristate 13-acetate and acetyl-beta-methylcholine. J Biol Chem 280:23559-65
Kamatchi, Ganesan L; Franke, Ruthie; Lynch 3rd, Carl et al. (2004) Identification of sites responsible for potentiation of type 2.3 calcium currents by acetyl-beta-methylcholine. J Biol Chem 279:4102-9