2008 |
Kirichok, Yuriy |
DP2Activity Code Description: To support highly innovative research projects by new investigators in all areas of biomedical and behavioral research. |
Molecular Biophysics of Mitochondrial Membranes: Defining Future Therapeutic Targ @ University of California San Francisco
Molecular Biophysics of Mitochondrial Membranes: Defining Future Therapeutic Targets Summary Mitochondrial dysfunction is implicated in several devastating diseases, such as neurodegeneration, obesity, diabetes, and cancer. Pharmacological interventions at the level of mitochondria can become an effective way to treat these pathological conditions. However, the development of such therapeutic tools is prevented by our incomplete understanding of the molecular mechanisms that underlie major mitochondrial functions, including energy production, setting the pace of aging, and controlling cell death. The transport of ions and molecules across the mitochondrial membranes is the foundation of the mitochondrial physiology and a lack of direct methods to study mitochondrial transmembrane transport is likely the most significant barrier to a better understanding of mitochondria. The key mitochondrial transport proteins, such as ATP synthase, the electron transport chain, and ion channels of the inner and outer mitochondrial membranes, could be best studied using the patch-clamp technique. This method revolutionized our understanding of ion channels and electrogenic transporters of the plasma membrane; however, an analogous application of the patch-clamp technique to mitochondria has been extremely difficult due to their small size and double- membrane architecture. Here we propose to develop an easily reproducible method for the application of the patch-clamp technique to both the inner and outer mitochondrial membranes for routine use in mitochondrial research. We will then apply the whole-membrane and single-channel modes of the patch- clamp technique to identify the full complement of ion channels and electrogenic transporters that are present in the inner and outer mitochondrial membranes. The accomplishment of these aims will provide an unparalleled functional essay for the key mitochondrial transport proteins, which, when combined with molecular biology, genetics, and protein crystallography, will facilitate significant advances in our understanding of the molecular workings of mitochondria and the subsequent development of therapeutic tools that control mitochondrial functions.
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0.915 |
2011 — 2015 |
Kirichok, Yuriy |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms That Control Ca2+ Signaling in Human Spermatozoa @ University of California, San Francisco
DESCRIPTION (provided by applicant): Intracellular Ca2+ and pH are two key regulators of the ability of sperm to fertilize an egg. Intracellular Ca2+ and pH are controlled in turn by sperm ion channels. Therefore, to understand the molecular mechanisms that control sperm function and male fertility, we need a more thorough understanding of sperm ion channels. Unfortunately, extreme difficulty in applying the patch-clamp technique to sperm cells has hampered our understanding of sperm ion channels and the molecular mechanisms controlling male fertility. We have overcome this barrier and developed a method to apply the whole-cell patch clamp technique to mouse and human spermatozoa. Surprisingly, our patch-clamp experiments revealed significant differences between ion channels in mouse and human spermatozoa. These differences indicate the potential pitfalls of relying on animal models for studying human male fertility and support the need to study these ion channels specifically in human sperm cells. Our long-term objective is to elucidate the mechanisms of ion channel-based signaling that control fertility in human spermatozoa. Here we propose three specific aims to expand our knowledge of sperm ion channels. In Specific Aim 1, we will identify the physiological regulators of human CatSper and Hv1 channels. We hypothesize that key regulators of sperm activity, such as progesterone, prostaglandins, cholesterol, and cAMP, are likely to mediate their actions on human spermatozoa by regulating CatSper or Hv1 channels. We will use the patch-clamp technique to test the effects of the above mentioned compounds on currents mediated by CatSper and Hv1 channels. In Specific Aim 2, we will identify the membrane (non- genomic) progesterone receptor of human spermatozoa. Our preliminary results have identified a narrow group of specific proteins as candidates for the sperm progesterone receptor. We will determine which of the candidate proteins serves as a progesterone receptor and will identify its ligand-binding domain for progesterone. In Specific Aim 3, we will characterize the acrosomal ion channels of human spermatozoa. We will develop a method for applying the patch-clamp technique to the acrosome of human spermatozoa and then use this method to characterize acrosomal Ca2+ channels that are likely to release Ca2+ from the acrosome and to identify the mechanisms that regulate their activity. The knowledge gained from this research will help us to understand the causes of male infertility and to develop new approaches for infertility treatment as well as contraception.
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0.915 |
2015 — 2018 |
Kirichok, Yuriy |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mitochondrial Uncoupling and Thermogenesis in Adipose Tissues @ University of California, San Francisco
In mammals, two tissues are primarily responsible for adaptive thermogenesis: brown fat and beige fat. Although brown fat has long been known as a specialized thermogenic tissue, beige adipocytes, a morphologically distinct type of fat cells that develop within white fat depots upon exposure to cold or high-fat diet, have been discovered only recently. Similar to brown adipocytes, beige adipocytes contain abundant mitochondria and seem to express mitochondrial uncoupling protein 1 (UCP1), which promotes mitochondrial production of heat by increasing the passive proton (H+) leak of the inner mitochondrial membrane. However, the mechanisms of mitochondrial uncoupling and thermogenesis in various beige fat depots as well as the relative contribution of UCP1 remain unclear. Moreover, brown and beige adipocytes retain partial thermogenic capacity even in UCP1-deficient mice. Therefore, this proposal is focused on the identification and characterization of UCP1-dependent and UCP1-independent mechanisms of thermogenesis in brown and beige fat. To this end, we developed a technique for direct patch-clamp recording from vesicles of the whole intact inner mitochondrial membrane (mitoplasts), which for the first time allowed high-resolution functional analysis of mitochondrial ion channels and transporters in their native membrane environment. Using this method, we have succeeded in recording thermogenic UCP1-dependent H+ leak currents across the inner mitochondrial membrane of brown and beige fat. Interestingly, not all beige fat depots possess this UCP1- dependent mitochondrial H+ leak, and using UCP1-deficient mice we also have discovered a novel UCP1- independent mechanism of mitochondrial uncoupling and thermogenesis in both brown and beige adipocytes. This proposal has a single specific aim: to characterize the mechanisms of mitochondrial uncoupling and thermogenesis in adipose tissues. Because adaptive thermogenesis in brown and beige fat consumes large amounts of energy from fat depots and reduces body adiposity and weight, this project will aid the development of therapeutic interventions to control obesity and diabetes.
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0.915 |
2017 — 2020 |
Kirichok, Yuriy |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms of Mitochondrial Uncoupling and Thermogenesis @ University of California, San Francisco
Project Summary Through substrate oxidation, mitochondria generate a high potential across the inner mitochondrial membrane (IMM). The energy of this potential is usually converted into ATP by the mitochondrial ATP synthase, but a fraction is dissipated as heat due to the presence of a H+ leak across the IMM. This mitochondrial H+ leak is mediated by specialized proteins of the IMM, such as uncoupling protein 1 (UCP1), and it has important physiological functions. It controls the metabolic efficiency of the body, helps to support the core body temperature, and reduces mitochondrial production of reactive oxygen species to protect against oxidative damage. The mitochondrial H+ leak is considered to be important in protective mechanisms against obesity, diabetes, and age-related degenerative disorders as well as against pathological conditions involving mitochondrial oxidative stress such as ischemia-reperfusion. Despite its physiological and pathophysiological significance, the mitochondrial H+ leak remains poorly understood, primarily due to the lack of direct methods to study it. We recently developed a method that removes this technical barrier and for the first time allows direct patch-clamp recording of H+ leak currents from the whole IMM. This method helped us resolve long- standing questions about the mechanism of the UCP1-dependent thermogenic H+ leak across the IMM of brown fat. In this application, we propose to use the patch-clamp technique to further characterize the mitochondrial H+ leak in several tissues that play important roles in thermogenesis and energy metabolism. The specific aims of this proposal are to: 1) identify the protein(s) responsible for the mitochondrial H+ leak in non-adipose tissues; 2) characterize the mechanism of the fatty acid-activated mitochondrial H+ leak via the adenine nucleotide translocator (ANT); 3) characterize the mechanism of the mitochondrial H+ leak induced by protonophores DNP and FCCP. Accomplishment of these specific aims will help us elucidate the principal mechanism that regulates metabolic efficiency and thermogenesis, and such knowledge will aid the development of therapeutic interventions to control obesity, diabetes, and age-related degenerative disorders.
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0.915 |
2019 |
Kirichok, Yuriy |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms of Mitochondrial Calcium Uptake @ University of California, San Francisco
Project Summary Mitochondrial dysfunction is implicated in several devastating diseases such as heart failure, ischemia- reperfusion injury, diabetes, neurodegeneration, and cancer. Thus, pharmacological interventions targeting mitochondria could become effective strategies for treating these pathological conditions. However, the development of such therapeutic tools is hindered by our incomplete understanding of the molecular mechanisms underlying major mitochondrial functions, including energy production, control of the pace of aging, and control of cell death. Mitochondrial Ca2+ transport dynamically regulates energy production in the heart depending on the energetic demand. This is accomplished by direct Ca2+ binding and activation of key enzymes involved in energy production. However, excessive mitochondrial Ca2+ influx such as that following Ca2+ overload in heart failure can lead to mitochondrial dysfunction and cardiomyocyte death via activation of the mitochondrial permeability transition pore (PTP). The mitochondrial calcium uniporter (MCU) is the principal channel responsible for mitochondrial Ca2+ uptake when the cytosolic Ca2+ level is elevated. MCU is a complex composed a central ion conduction pore and several regulatory subunits. Despite the clear significance of MCU in mitochondrial and heart physiology, rigorous biophysical analyses using direct patch-clamp recording of MCU currents to study the molecular components of the MCU complex have not been achieved. Here we develop the first heterologous expression system for patch-clamp based structure-function analysis of MCU in the inner mitochondrial membrane (IMM). This system employs both whole-IMM and single-channel modes of the patch-clamp technique to study mutants of the MCU pore and auxiliary subunits to enable a comprehensive study of structural features that mediate MCU-complex selectivity, activation, and physiological regulation. We plan to answer two central questions in the field: 1) How does the MCU pore allow selective one-way Ca2+ transport into mitochondria in the presence of much higher concentrations of monovalent cations? 2) How do the auxiliary subunits control MCU gating to achieve precise control over ATP production without causing excessive mitochondrial depolarization or MPT? Answering these questions will provide an essential framework for the development of pharmacological interventions to control MCU activity and mitochondrial function under physiological and pathophysiological conditions.
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0.915 |
2020 — 2021 |
Kirichok, Yuriy |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Molecular Biophysics of Mitochondrial Membranes @ University of California, San Francisco
Project Summary This project is focused on understanding of the molecular mechanisms that control energy conversion within the cell power plant, the mitochondrion. Transport of ion and metabolites across the inner mitochondrial membrane is the foundation of mitochondrial physiology. Here we study the molecular mechanisms involved in passive uptake of H+ (?mitochondrial H+ leak?) and Ca2+ (?mitochondrial Ca2+ uptake?) into mitochondria down the negative voltage (??) across the inner mitochondrial membrane. The mitochondrial H+ leak is responsible for the conversion of chemical energy of mitochondrial substrates into heat. The mitochondrial Ca2+ uptake lets Ca2+ enter mitochondria during cytosolic Ca2+ transients to stimulate Ca2+-dependent enzymes of the Krebs cycle and mitochondrial energy conversion. The mitochondrial H+ leak and Ca2+ uptake are mediated by specialized integral proteins of the inner mitochondrial membrane called uncoupling proteins (UCPs) and mitochondrial Ca2+ uniporter (MCU) correspondingly. The molecular and functional characterization of UCPs and MCU has been difficult due to the inability to measure H+ and Ca2+ currents across the inner mitochondrial membrane directly. We have resolved this major technical barrier and developed a method for direct patch- clamp recording of UCP and MCU currents across the whole inner mitochondrial membrane. Using this method, we propose to address the structure-function relations within UCPs and MCU to understand the molecular mechanisms that govern H+ and Ca2+ translocation via these membrane transport proteins. For UCPs, we plan to identify the mechanisms by which free fatty acids, the endogenous UCPs activators, cause H+ translocation via UCPs. We also propose to develop a new generation of drugs that can activate H+ leak via UCP and be used for the treatment of type II diabetes, obesity, and fatty liver. For MCU, we propose to reveal the molecular mechanisms responsible for the exceptionally high Ca2+ selectivity of MCU, the MCU inward rectification (one-way permeation of Ca2+ into mitochondria), and the MCU potentiation by cytosolic Ca2+. This research will resolve several long-standing problems in the field of bioenergetics and will eventually enable pharmacological control of key mitochondrial functions in therapeutic and research purposes.
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0.915 |