AMP-ACTIVATED PROTEIN KINASE - THE KEY SENSOR OF CELLULAR ENERGY CHARGE D. Grahame Hardle, Dundek University, Scotland

The AMP-activated protein kinase (AMPK) is the downstream component of a protein kiqase cascade that is activated b energy depletion, signalled b a rise in AMP coup ed with a fall in ATP, and/or a. fa I I-I Y ‘(.

P hosphocreatine [l . Falling. energy cha

ll he system h an u rasensltive manner. T- eh;cWaIvai

then switches off ATP-consumin

l] Hardie, D.G., Carling, D. and Carlson, M. (1998) Ann. Rev. Biochem. 67, 821-855.

[2] Kudo, N., Gillespie, J.G., Kung, L., Witters, L.A., Schulz. R., Clanachan, AS. and Lopaschuk, G.D. (1996) B&him Biophys Acta 1301, 67-75.

[3] Marsin, A.S. et al. (2000) Curr Biol 10, 1247-1255.

STUDIES ON AMP-ACTIVATED P,ROTEIN KINASE USING A MOUSE MUSCLE CELL LINE Davld Carling, Cellular Stress Group, MRC Clinical

Sciences Centre, London, UK.

The AMP-activated protein kinase (AMPK) is activated by stresses which deplete ATP. causing a concomitant rise in AMP. Once activated, AMPK initiates a series of

responses aimed at restoring the energy balance within the cell. AMPK is a heterotrimer composed of an cx catalytic

subunit and p and y regulatory subunits. lsoforms of all

three subunits have been identified, although their physiological significance remains enigmatic. We have begun to characterize the in viva expression of the different isoforms as a first step towards elucidating their precise function. Our results, together with the recent finding that a mutation within the y3 isoform results in excess glycogen

storage in skeletal muscle, suggest that the y subunit plays

an important role in regulating the kinase. In addition, we have been studying the activation of AMPK and its role in the regulation of glucose uptake in a mouse muscle cell line derived from the H-2K-tsA58 transgenic mouse. Using this model system we have started to delineate the mechanism of activation of AMPK in response to different stress treatments and to study the pathway by which AMPK increases glucose uptake in muscle.

Coupling cellular energetics with membrane excitability: A role for phosphotransfer reactions Andre Terzic, Mayo Clinic, Mayo Foundation, Rochester, MN, USA

Efficient communication between cellular energetics and membrane metabolic sensors is required for regulation of cell excitability. Plasmalemmal K-ATP channels are unique nucleotide sensors of the cellular metabolic state that adjust membrane potential in response to intracellular metabolic oscillations. However, the mechanism that facilitates nucleotide exchange in the K-ATP channel environment, and promotes coupling of membrane electrical events with cellular metabolic pathways remains unknown. Here, we demonstrate that intracellular phosphotransfer relays regulate the K-ATP channel response to metabolic challenge, promoting delivery of mitochondria generated signals to the channel site. Such a signal processing function for phosphotransfer reactions was lost in cells lacking phosphotransfer enzymes, thereby disrupting K-ATP channel coupling from cellular metabolism. In this way, intracellular phosphorelays provide novel pathways for integration of cellular energetics with membrane electrical events.

MITOCHONDRIAL KATP CHANNELS: NOVEL TARGETS FOR MODULATING FUNCTION OF MYOCARDIAL AND VASCULAR CELLS Brian O’Rourke*, Medicine, Johns Hopkins University, Baltimore, MD, USA

Mitochondrial KATP channels (mitoKATP) have recently emerged as important triggers and effecters of cellular urotection against ischemia and reperfusion injury. ks such, growing interest has focuseh on the molecular identitv of mitoKATP. the isoform suecificitv of K channel mo&lators, and thk mechanisms by which mitoKATP channels confer protection. Recent investigations from our group have indicated that the mitoKATP isofonn is distinct from its congeners on the surface membrane. Adenoviruses were constructed to specifically suppress the function of either Kir6.1 or Kir6.2 in a dominant negative manner and myocytes were infected for 48-72 hrs. These viruses effectively and selectively suppressed surface membrane KATP channels; however, there was no effect on the function of mitoKATP, as determined using a mitochondrial flavoprotein fluorescence assay. Although mitoKATP differs from the known cloned KATP channel isoforms, it appears to share a similar pharmacological profile with Kir6.WURl. The mechanistic actions of mitoKATP opening are currently controversial. Recent data from our group suggests that a primary effect of mitoKATP activation is to suppress mitochondrial Ca overload associated with metabolic inhibition. Furthermore, mitoKATP openers are effective inhibitors of apoptosis induced by oxidative stress by preventing the activation of the mitochondrial permeability transition and cytochrome C release.

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