Toshinori Hoshi - US grants

Potassium channels are universally found in biological systems and play major roles in the cellular signal transduction process. The generally decrease the membrane excitability by clamping the membrane voltage near the potassium equilibrium voltage. Functional properties of the potassium channels can ultimately influence how the organism responds to its environment. Several different potassium channels have been characterized. Inward-rectifier potassium channels open in response to hyperpolarization and they are involved in maintaining normal resting potential. By influencing the resting potential, these inward-rectifier channels regulate the cellular excitability. Fore example, in heart muscle, the inward-rectifier channels greatly influence the rhythmicity, effectiveness of various antiarrhythmic agents, and the action potential conduction. The proposed research focuses on the cloned inward-rectifier potassium channel (KAT1) expressed in a heterologous expression system. The goal of the research is to understand how this channel opens and closes at the molecular level by developing a kinetic model to describe the behavior. The channel protein structure will be rationally mutated and the alterations in the function will be quantitatively assayed using electrophysiological methods. the preliminary results indicate that the opening of the KAT1 channel requires both hyperpolarization and some intracellular factors. The proposed research will identify the intracellular factors and investigate which amino acid residues of the channel involved in mediating the effects of these intracellular factors. The research proposed here will also investigate how the KAT1 channel opens in response to hyperpolarization. This research will examine if the structural and functional properties of the activation of the KAT1 channel favored by hyperpolarization are similar to those of the depolarization-activated potassium channels. By examining the molecular and biophysical properties to the KAT1 channel activated by hyperpolarization and by comparing the results with those obtained from depolarization-activated potassium channels, it should be possible to gain insights into the universal molecular principles involved in the voltage-dependent channel behavior. Furthermore, since the KAT1 channel is activated by the intracellular agonists and hyperpolarization, the results obtained will also be very relevant to the studies on various agonist-activated ion channels.

DESCRIPTION: Previous studies have shown that, like many other proteins, amino acid residues of potassium channels are subject to oxidation by reactive oxyge species. Oxidation of amino acid residues, especially of cysteine residues, is well documented. Methionine can be readily oxidized to form methionine sulfoxide. Oxidation of methionine is unique in that it is reversible and that reduction of oxidized methionine requires an enzyme, methionine sulfoxide reductase (MsrA). The reversibility of methionine oxidation catalyzed by MsrA suggests that it could act as an important cellular regulatory mechanism. Preliminary results indeed suggest that oxidation of methionine residues in Shaker potassium channels has dramatic effects on the channel activation and deactivation. This project will establish the dynamic functional role of methionine oxidation as a key player in regulation of cellular excitability. Shaker potassium channels will be expressed in Xenopus oocytes and their macroscopic and single-channel current properties examined using the two-electrode voltage clamp and patch-clamp methods. The effects of methionine mutations and MsrA co-expression will be quantitatively assayed to elucidate the biophysical mechanisms involved. The importance of methionine oxidation an its reversal by MsrA in a variety of other potassium channels and voltage-dependent calcium channels will also be examined. Results from the proposed research will establish a novel cellular excitability mechanism involving methionine oxidation and reduction.

Apoptosis or programmed cell death can be induced by a variety of stimuli. Apoptosis is considered to be involved in many physiological and clinical phenomena, including ischemic injury, many forms of cancer, and neurodegenerative diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, and some prion-related disorders. Many apoptosis-inducing stimuli and gene products have been identified. The molecular mechanisms by which these stimuli trigger the cell-death program are, however, only poorly understood. The research program proposed here will focus on how an apoptosis-trigger gene, reaper, promotes cell death in electrically excitable cells. The reaper gene is expressed in cells destined to undergo apoptosis 1 to 2 hours before the cell death program starts. A sequence comparison of the N-termini of Reaper and a "A-type" voltage-dependent potassium channel (ShB) shows that the Reaper protein could work as an inactivation ball to block potassium channels. Our preliminary results indeed show that the full-length Reaper protein blocks voltage-dependent potassium channels by inducing very stable inactivation. Our preliminary results also show that Reaper slows down the inactivation process of voltage-dependent sodium channels. The proposed research program will examine how the Reaper protein alters functional properties of voltage-dependent ion channels to promote apoptosis. The central hypothesis is that Reaper alters properties of the plasma membrane ion channels to promote depolarization and that this depolarization facilitates apoptosis. We will test this hypothesis using heterologously expressed ion channels in Xenopus oocytes as well as native mammalian channels. We will examine how induced expression Reaper affects the ion channel properties and membrane potential in mammalian cells. The results expected from the proposed research will establish how some apoptosis-trigger proteins promote cell-death and the results may be useful in designing therapeutic interventions to treat the diseases that involve apoptosis.

Oxidative damages to cellular constituents are postulated to accelerate aging and shorten the life-span of an organism. Aging in many species is accompanied by declines in physical activity and reproductive vigor. A variety of strategies to minimize oxidative damages to increase the life- span have been proposed. The amino acid methionine is easily oxidized and the repair of oxidized methionine is catalyzed by the enzyme peptide methionine sulfoxide reductase (MSRA). Here we will examine life-span extension roles of the anti-oxidant MSRA in the model organism Drosophila. Previous results suggested that constitutive over-expression of MSRA throughout the entire Drosophila life markedly extended the life-span. The study proposed here will test whether it is possible to extend the life-span and delay senescence-induced declines in the physical activity level and reproductive capacity if MSRA is over- expressed after the animal reaches adulthood. Temporally-controlled over-expression of MSRA is achieved using the RU486-dependent "GeneSwitch" protocol. The effects of induced MSRA over-expression on the survival distribution, oxidative stress resistance, physical activity and reproductive behavior are examined. The results expected will provide insights into the mechanism underlying the life-extension effect of MSRA and implicate MSRA a possible therapeutic agent to slow aging and senescence-induced changes in physiology and behavior.

DESCRIPTION (provided by applicant): Oxidation of amino acids by reactive oxygen species is considered to accelerate aging. The amino acid methionine is readily oxidized to form two epimers of methionine sulfoxide, methionine-R-sulfoxide (met-R-O) and methionine-S-sulfoxide (met-S-O). The enzyme methionine sulfoxide reductase A (MSRA) reduces met-S-O while the enzyme methionine sulfoxide reductase B (MSRB) reduces met-R-O. Multiple forms of MSRA and MSRB that differ in tissue and subcellular distributions are now known. Increasing evidence suggests that methionine oxidation may be an important determinant of the time course of normal aging. However, there is no systematic information available how the met-R/S-contents and MSRA/B activities change with age. Furthermore, it is not known whether different MSR forms play similar roles in aging. Using the model organism Drosophila, the study proposed here will provide a comprehensive view of the roles of methionine oxidation and MSRs in normal biology of aging. Age-dependent changes in the oxidized methionine contents and MSR activities in different tissues and subcellular fractions are systematically measured. Different forms of MSRs are overexressed in different tissues and subcellular regions to compare their efficacies in lifespan extension and delaying the onset of decline in the physical activity level and reproductive vigor. Furthermore, gene expression profiles in the long-living MSRA flies are systematically compared with those of control flies. The study will also test whether a food supplement, S-methyI-L-cysteine, could act to extend the animal lifespan by participating in the reversible methionine oxidation cycle involving the MSR system. The results expected should prove valuable in designing interventions to extend lifespan.

DESCRIPTION (provided by applicant): Many of us will be eventually afflicted with vascular disorders, such as hypertension and brain hemorrhage. Cerebral hemorrhage is often followed by delayed cerebral vasospasm, which itself represents a significant morbidity and mortality factor. Cerebral vasospasm is characterized by a long-lasting abnormal contraction of vascular smooth muscle cells. Previous studies have suggested that blood breakdown products, such as heme, CO, bilirubin and bilirubin oxidation end products (BOXes), may be involved but the underlying mechanism has remained elusive. Among the many proteins involved in regulation of vascular smooth muscles, large-conductance calcium- and voltage-activated (Slo1 BK) potassium channels play a critical role in vascular relaxation. The importance of Slo1 channels in regulation of vascular tone suggests that many disorders of vascular relaxation, including cerebral vasospasm, may involve dysregulation of Slo1 channels. Because bilirubin and bilirubin oxidation end products are implicated in vasospasm and Slo1 channels play a critical role in regulation of vascular tone, we hypothesize that heme catabolic products may contribute to cerebral vasospasm by modulating Slo1 channels. To test this hypothesis, effects of heme catabolic products (CO, bilirubin and bilirubin oxidation end products) on the Slo1 channel function are electrophysiologically and quantitatively investigated using macroscopic and single-channel ionic current and macroscopic gating current measurements. These biophysical measurements are complemented with physiological isometric force measurements using aortic blood vessels from wild-type and Slo1 knockout mice. The results expected from the research program proposed will establish a new paradigm of CO sensing by Slo1 and will highlight Slo1 channels as an important causal link between cerebral hemorrhage and cerebral vasospasm. These novel conceptual frameworks in turn suggest new therapeutic strategies for disorders of vascular relaxation including delayed cerebral vasospasm.

DESCRIPTION (provided by applicant): Large-conductance Ca2+- and voltage-gated K+ (Slo1 BK) channels play numerous physiological and pathophysiological roles and their allosteric gating mechanism is subject to modulation by a variety of cellular signaling pathways. Increasing evidence suggests that certain lipids may serve as signaling molecules. We propose to reveal the biophysical and physicochemical mechanisms of modulation of Slo1 BK channels by two lipid messengers, phosphatidylinositol 4,5-bisphosphate (PIP2) and docosahexaenoic acid (DHA), an omega-3 long-chain polyunsaturated fatty acid enriched in oily fish. The effect of PIP2 on the Slo1 BK channel was reported recently but the mechanism is only poorly known. We will fill this critical knowledge gap by performing thorough mechanistic electrophysiological measurements. Furthermore, we will identify the structural determinants of the auxiliary beta subunit important for the action of PIP2. Our electrophysiological measurements will be complemented with measurements of tryptophan fluorescence of the isolate and purified Slo1 gating ring protein. The biophysical and physicochemical mechanisms of the action of DHA, an emerging lipid messenger, on the Slo1 channel will be also investigated similarly using the electrophysiological and fluorescence methods. The research outcome is expected to provide definitive mechanisms of the PIP and DHA actions on the allosteric gating mechanism of the Slo1 channel and establish the novel paradigm that the Slo1 channel is an omega-3 fatty acid receptor. The modulation of the Slo1 BK channel by DHA may underlie the health-promoting effects of omega-3 long-chain fatty acids.

Project Summary Long-chain polyunsaturated omega-3 fatty acids, such as docosahexaenoic acid (DHA) with a 22-carbon chain, are found abundantly in oily fish including anchovy, herring, mackerel, and salmon. These omega-3 fatty acids are widely thought to have multiple health-promoting effects. Evidence suggests that DHA decreases blood pressure, especially in hypertensive patients. We hypothesize that the hypotensive action of DHA is mediated by its stimulatory effect on large-conductance calcium and voltage-gated potassium (Slo1 BK) channels important in blood pressure regulation. The research program proposed here will provide molecular and atomic basis of the hypotensive action of DHA involving Slo1 BK channels using a variety of methods. We postulate that a hydrogen bond between a tyrosine residue in the S6 segment of the channel and the carboxylate group is critical in destabilizing the closed conformation of the ion conduction gate and this interaction underlies the hypertensive action. Using the physicochemical principles elucidated, we will rationally design, synthesize and test fatty-acid activators of Slo1 BK channels. The anticipated outcome of the research program has potential to explain blood pressure regulation based on the hydrogen bonds formed between specific tyrosine residues of the Slo1 BK channel and DHA and provide a solid mechanistic foundation for discovery and development of pharmaceuticals and nutraceuticals for blood pressure management.