AMINO ACID PERMEASE INVOLVEMENT IN THE VOLATILE ANESTHETIC RESPONSE OF SACCHAROMYCES CEREVISIAE

Open Access
Author:
Keasey, Nikki R
Graduate Program:
Biochemistry and Molecular Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
December 11, 2007
Committee Members:
  • Ralph Lauren Keil, Committee Chair
  • Jong Kak Yun, Committee Member
  • Leonard Shelton Jefferson Jr., Committee Member
  • Laura Carrel, Committee Member
  • Ross Shiman, Committee Member
  • Scot R Kimball, Committee Member
Keywords:
  • yeast
  • volatile anesthetics
  • nutrient transport
Abstract:
Despite the clinical importance of volatile anesthetics, their mechanisms of action remain unknown. Understanding how anesthetics produce their numerous cellular effects will facilitate the design of safer anesthetics with fewer undesirable side effects. The yeast Saccharomyces cerevisiae is being used as a model to elucidate anesthetic actions. Studies in yeast show that availability of specific amino acids plays a key role in the response of this organism to anesthetics. The volatile anesthetic isoflurane inhibits the uptake of leucine and tryptophan, inducing a starvation response and growth arrest in appropriately auxotrophic strains. Overexpression of TAT1, TAT2, or BAP2 genes encoding permeases that import these amino acids, renders such strains resistant to volatile anesthetics. Conversely, deletion of these genes leads to increased sensitivity to anesthetics. These findings implicate specific amino acid permeases as candidate targets of anesthetics. The high-affinity tryptophan transporter Tat2p, which is encoded by TAT2, was inhibited in a time- and dose-dependent manner during isoflurane exposure. This inhibition was rapid and reversible. Amino acid uptake studies revealed that the inhibition of Tat2p by isoflurane was not competitive, suggesting that anesthetics do not affect the ability of Tat2p to bind tryptophan. Inhibition of transport was not due to degradation or this permease or relocalization away from the plasma membrane. These findings are consistent with a model where isoflurane decreases Tat2p transporter activity, either through a direct interaction or a membrane-mediated effect. Mutations within TAT2 that render cells resistant to isoflurane were isolated. Characterization of these mutants revealed that anesthetic resistance can result from an overall increase in tryptophan uptake in the presence and absence of drug, a decrease in the inhibitory effects of anesthetic on uptake, or a combination of these two factors. Tat2p provides a model of anesthetic effects on a candidate drug target in a single-celled organism. A better understanding of the effects of volatile anesthetics on amino acid permeases will provide insight into the mechanisms of action of these clinically essential drugs.