Ion channels use the energy stored in ionic gradients to initiate rapid signaling events essential for the function of virtually every cell in the body. Although channel activation can occur in response to a variety of different stimuli, once open all channels share a common feature--they allow millions of ions per second to cross the membrane. Pump proteins on the other hand, use the energy in ATP to establish the ionic gradients necessary for channel function. Pumps generally move hundreds of ions per second, and hence their density in the membrane is by necessity much higher than that of channels. These striking differences led to the view that pumps and channels move ions across membranes by very different mechanisms. However, recent studies on the potent marine toxin, palytoxin (PTX), challenge this concept. PTX binds with picomolar affinity to the Na+,K+-ATPase (NKA) and converts the pump into a non-selective cation channel. Evaluation of PTX action suggests that the fundamental difference between channels and pumps resides not so much in the molecular architecture of the ion translocation pathway itself, by rather in the intrinsic gating properties of the protein. More importantly, the fact that high affinity toxins can induce a channel-mode of pump operation suggests that endogenous mechanisms may also exist that lead to the same channel mode and which may have either an important physiological role in cell signaling, or produce disastrous consequences for cell function and survival if not adequately controlled. Our preliminary studies have shown that another marine toxin called maitotoxin (MTX), converts the plasmalemmal Ca2+-ATPase pump (PMCA) into a Ca2+-permeable, nonselective cation channel which ultimately causes Ca2+-overload induced necrotic cell death. Furthermore, we discovered that the Ca2+ channels activated by MTX in vascular endothelial cells are biophysically identical to the channels activated by the model oxidant, tert-butyl-hydroperoxide or by oxidized glutathione (GSSG). Thus, changes in cellular redox status, appears to trigger conversion of the PMCA pump into a channel. Furthermore, our recent studies showed that the PMCA can be directly glutathionylated both in vitro and in vivo, in response to oxidant stress. Therefore, in Specific Aim #1, we will test the hypothesis that oxidative stress converts the PMCA pump into a non-selective cation channel, and in Specific Aim #2, we will determine the role of glutathionylation in both inhibition of PMCA catalytic activity and in the pump-to-channel conversion. The conversion of the PMCA pump into a channel provides a novel and ubiquitous mechanism by which oxidative stress initiates Ca2+-overload and provides a new molecular target for therapeutic intervention in a variety of pathological conditions including atherosclerosis, ischemia-reperfusion injury, Alzheimer's disease, and biological aging.
Oxidative stress is thought to play a role in the development or progression of a variety of diseases including hardening of the arteries (atherosclerosis), heart attack (myocardial infarction), stroke, Alzheimer's disease, and even biological aging. A rise in cellular calcium during oxidative stress is thought to be one of the earliest detectable events leading ultimately to cell death. The experiments proposed herein, will test an entirely new model for how this rise in calcium occurs and provide a new and important molecular target for therapeutic intervention.
