Enrico Stefani - US grants

The proposed research project will be concerned on the biophysical properties of voltage-gated Ca channels in rat and frog skeletal muscle fibers. These studies are designed to define the physiological role of voltage-dependent Ca entry for muscle contraction. Previous studies (1 to 11) have described a voltage-dependent Ca entry in frog skeletal muscle fibers. These Ca channels are located in the tubular system and its role during muscle contraction is not clearly established. We plan to study, using the three microelectrode voltage clamp technique, the loose patch clamp technique and single channel recordings, the kinetic properties, permeation mechanisms, modulation and differentiation of the slow Ca channels. Moreover, the distribution of intracellular Ca during Ca current will be also investigated. In addition to this, some preliminary data indicate the existence of a fast Ca channel in muscle fibers. This preliminary finding will be carefully investigated in relation to the possible Ca entry during a single action potential or tetanic stimulation. An accurate description of the gating process, modulation and differentiation of Ca channels will be the basis to plan future mechanical experiments to determine the physiological role of voltage-dependent calcium entry.

The long term objective of the proposed research project is to define the functional role of voltage gated Ca++ entry in skeletal muscle. Voltage and current clamp experiments will be carried out in isolated skeletal muscle from the frog with the cut fiber preparation in the vaseline gap technique. Myoplasmic Ca++ transients will be optically measured with Antipyrylazo III. Ionic currents, charge movement and changes in intracellular Ca++ concentration will be studied simultaneously. The slow Ca++ channel is located in the tubular system and it is too slow to be activated during a single action potential, however this is not the case for the recently described fast Ca++ channel. In initial experiments the effect of reducing external Ca++ on the Ca++ transient associated with the action potential will be investigated. In order to distinguish the activation of these channel in relation to charge movement and Ca++ transients, the pharmacology and modulation of fast and slow Ca++ channels will be studied in detail. In addition the recent described fast Ca++ channel will be further characterize in term of its localization, pharmacology and kinetic. These studies will be performed in a comparative way in preparations with variations of Ca++ channel currents such as the denervated muscle fiber, the end-plate region and the tail muscle from tadpoles. Ca++ channels were recently described in skeletal tonic fibers, where Ca++ entry may play a role in contraction. This point will be clarified by simultaneously measuring Ca++ currents, Ca++ transient and charge movement. An insight on the role of Ca++ channel activation will be obtained also from developmental studies, with measurements of Ca++ macroscopic and single currents that will be performed in primary cultures of skeletal muscle of new born rats. To define the physiological role of voltage gated Ca++ entry in skeletal muscle is a relevant problem in the medical sciences, since, Ca++ channel alterations have been recently described in muscle diseases such as muscular dysgenesis and muscle dystrophia.

The long term objective of the present proposal is to define the physiological role of myometrial ionic channels in relation to the excitability and contractile function of the uterus. We propose that ionic channels expression and their regulatory mechanism(s) are influenced by hormones. Single cells freshly dissociated or maintained in tissue culture and purified plasmalemma membrane fractions from uterine smooth muscle will be used. Freshly dissociated cells will reflect the hormonal condition of the animal while primary culture will be used as a model to study the action of hormones. Macroscopic currents will be explored using both the whole cell patch clamp and the intracellular microelectrode voltage clamp techniques. Single channels will be made in conjunction with binding studies of adrenergic and dihydropyridine ligands to determine changes in number or type(s) of channels or receptors related to the hormonal conditions. In initial experiments macroscopic currents will be characterized in freshly dissociated cells from animals at diestrus. Our study will be mainly focused on voltage dependent K, Cl and Ca channels (voltage dependency, kinetics, pharmacology and regulation). Parallel studies will be performed on single channel in intact cells and in bilayers. Furthermore, the affinity and number of sites for adrenergic and dihydropyridine receptors will be measured. To test the hypothesis that the expression of ionic channels and their regulatory mechanism(s) may be affected by hormones, the next procedures will be followed: I. Animals at estrus and diestrus phases of the estrus cycle will be comparatively studied. Acutely dissociated cells and membrane vesicles incorporated into bilayers will be used. The characteristics of membrane K, Ca and Cl currents and the corresponding single channels will be analyzed. To explain the differences in activity of the uterus during these phases, the actions of adrenergic agents, oxytocin and prostaglandins on Ca and K channels and their mechanism(s) will be studied. These studies will be correlated with binding studies of adrenergic and dihydropyridine ligands; II. Hormonal changes will be induced in animals or in cultured cells. Ionic channels will be studied in acutely dissociated cells from: a) spayed animals treated with estradiol or progesterone; and b) in cultured cells from animals at estrus treated with estradiol or progesterone. Ionic channels from rats at mid- and late- pregnancy will be studied following the same experimental plan as for cycling animals. The knowledge of the functional role and regulation of ionic channels will allow a comprehension of the excitability of the uterus in relation to hormonal effects. This will be important in the medical sciences to determine the role of hormones in the reproductive cycle and to design or improve therapeutic treatment(s) for pathological situations such as premature labor and dysmenorrhea.

The long term goal of this proposal is to gain insight on how voltage dependent Ca2+ channels function in molecular terms by performing structure-function studies of cloned Ca2+ channels. The main focus is to study the movement of the voltage sensor (gating currents) and its coupling to channel opening and closing, and the mechanism(s) and the molecular domains for the regulatory actions of coexpressed beta subunits, of Ca2+ influx and of phosphorylation. These studies can be performed because the success in measuring gating currents of cloned Ca2+ channels expressed in frog oocytes. This is due to: 1) a large functional expression, 2) the fast response of the cut-open oocyte vaseline gap technique, and 3) the success of the giant-macropatch technique (20-30 mum) for noise analysis. The main questions are; 1. How are the open and closed states of the channel related to charge movement? 2. What are the number of effective charges per channel and the unitary charge element? 3. Can the coupling between the voltage sensor and the pore be probed with mutagenesis to define molecular domains for the charge movement directly associated to channel opening? 4. Is the inactivation process associated with charge immobilization? 5. Which are the mechanisms by which beta regulatory subunits and phosphorylation facilitate channel opening? 6. Can we identify critical phosphorylating sites for channel function? 7. Are these regulatory actions altering specific components of the gating currents or are they altering the final coupling between the charge movement and channel opening? The Specific Aims are: 1. To investigate the properties of Ca2+ channel charge movement to gain insight on the molecular mechanism(s) and the domains involved in the coupling between the movement of the voltage sensor and channel opening; 2. To define the charged residues and the structural determinants in the alpha1 pore subunit responsible for the voltage dependence of Ca2+ channel activation; 3. To study the mechanism(s) of Ca2+-dependent inactivation in the cardiac alpha1C channel, and of voltage- dependent inactivation in the neuronal alpha1E channel; 4. To define the mechanism(s) and molecular domains for the interactions between the Ca2+ channel alpha1 pore forming subunits and the regulatory beta subunits; and 5. To define in alpha1 subunits the role of phosphorylation in the coupling of the voltage sensor with channel opening. These structure- function studies of Ca2+ channels should serve as the basis for understanding the actions of many therapeutical agents anf for the development of new drugs.

The long term goals of this proposal are to gain insight on how voltage dependent ion channels function in molecular terms, and to define the different protein conformational changes associated with transmembrane voltage changes and channel gating. Ionic currents and gating currents will be measured. The gating currents are the electrical manifestation of the movement of the voltage sensor. The experiments are planned for cloned K channels. Two main preliminary findings are key aspects of this proposal: 1. the discovery of an early peak component of charge movement recorded at high bandwidth (200 kHz) and 2. a tendency of gating current rates to saturate at extreme potentials. The early component and the tendency to saturation with voltage of gating current rates are consistent with the view the channel transits a large number of states, like in a diffusion process of the charge along an energy landscape shaped with barriers and wells. The specific aims are: 1. To investigate the early component of gating currents. ON and OFF gating currents were preceded by a fast component. 2. To investigate the mechanism underlying the tendency of gating current rates to saturate with voltage. The gating current rates tend to saturate at high potentials. A model in which the charge diffuses along an energy landscape shaped with barriers and wells will be tested. 3. To gain insight on channel function via a characterization of the gating current noise and to define the charged residues involved. The properties of gating noise will be investigated. 4. To investigate mechanism(s) of slow inactivation by a characterization of ionic and gating current properties during long depolarizations. We will test the hypothesis that there are two types of slow inactivation and 5. To obtain a global model of Shaker K channel function from ionic and gating current data. These determinations, in conjunction with gating current noise measurements, should provide well defined constraints for the prediction of the conformational changes occurring during channel gating. This research on fundamental properties of ion channel should be valuable to the design and test therapeutic drugs.

The long term goal of this proposal is to unravel hormonal-regulated changes of ion channel expression and function in uterine smooth muscle, with special emphasis on Ca2+ dependent K+ channels (MaxiK), fast transient K+ channels (Kv4.3, ITO) and L-type Ca2+ channels. The main hypothesis is that during pregnancy differential expression of K+ and Ca2+ channel types, isoforms, and regulatory subunits contribute to the dramatic changes that occur in uterine excitability and contractility. Our preliminary data show that: 1) Expression of the MaxiK channel alpha subunit, the Kv4.3 K+ channel (ITO), and the Ca2+ channel alpha1C and beta2a subunits, varies during pregnancy; 2) RNAse protection assay (RPA) shows that the reduction in protein expression of MaxiK alpha subunit and Kv4.3 channels correlates with changes in mRNA levels; 3) Blockade of Kv4.3 channels enhances contractility; 4) A novel splice insert of the Maxi K alpha subunit may act as a dominant negative expression regulator; 5) Reduction in the expression level, at the end of pregnancy, of MaxiK and Kv4.3 channels may be associated with altered trafficking, and 6) Myometrium from non-pregnant rats primed with beta-estradiol have reduced Kv4.3 channel expression. Thus, the questions to answer are: a) What are the physiological and pharmacological changes that MaxiK channels undergo during pregnancy? b) Which splice variants of MaxiK alpha subunit are present in myometrium and what is their functional impact? c) What is the molecular nature of ITO currents in myometrium? d) What is the role of ITO currents in myometrial contractility? e) What are the molecular components of L-type Ca2+ channels (alpha1C and beta subunits), and are they differentially expressed during pregnancy? f) What are the functional properties of L-type Ca2+ currents at different stages of pregnancy? g) Which is the mechanism(s) responsible for the changes in expression levels of K+ and Ca2+ channels, and which sex hormone(s) controls channel expression? The Specific Aims will use a multidisciplinary approach to investigate at different stages of pregnancy and with hormonal treatment: 1) changes in function, protein expression and mRNA levels of the MaxiK channel, 2) which MaxiK alpha subunit splice variants are present in myometrium, their functional properties, and their expression, 3) the molecular nature and function of fast transients K+ currents, and 4) the nature and changes in alpha1C Ca2+ channels and regulatory beta subunits. These studies will be relevant to design or improve therapeutic treatment(s) for pathological situations such as premature labor and dysmenorrhea.

DESCRIPTION (provided by applicant) The main hypothesis of this proposal is that different types of K+ and Ca2+ channels have specific functions and they are expressed depending on the biological status of the nerve muscle synapse. The main questions are: What types of K+ and Ca2+ channels are expressed at the mature motoneuron and its nerve terminal junction? Is the functional expression of the Ca2+ channel dependent upon protein kinase or phosphatase activity? What are the changes in K+ and Ca2+ channel expression or modulation during development? We plan to circumscribe our efforts and concentrate on the expression and localization of the L-type voltage dependent Ca2+ channel (L-VDCC), the Ca2+ activated K+ channel (Kv,ca), and the fast transient K+ channel (Kv4.2 and Kv4.3). To answer these questions we plan to study the following: 1. To determine the expression and modulation of L-VDCCs at the mature neuromuscular junction (NMJ). 2. To investigate the functional role of Kv4.2, Kv4.3 and Kv,ca at the mature motor nerve terminal. 3. To determine developmental associated changes in the functional role of K+ and Ca2+ channel subtypes at the neonatal NMJ. 4. To determine the expression levels and localization of different types of K+ and Ca2+ channel subunits in developing motoneuron soma and nerve terminals. We will measure spontaneous and evoked TR in the presence of K+ and Ca2+ channel specific blockers in nerve-muscle preparations to determine their participation in the release process. Extracellular presynaptic currents will be recorded via a perineural inserted microelectrode This will allow us to gain direct information about channels expressed in the nerve terminal related and unrelated to TR. Specific antibodies for the a subunits and their regulatory b subunits will be used to determine their expression. These studies will provide basic information on synaptic physiology which is a key point to understand NMJ disorders

DESCRIPTION (provided by applicant): The long term goal is to unravel cellular and molecular mechanisms involved in cardiac-electrical and molecular-remodeling during functional hypertrophy in pregnancy, and recovery in postpartum; in particular, those related to K+ channel expression/function. I to K+ shapes the cardiac action potential duration in both rodents and humans. In failing hypertrophic hearts, I to-f molecular components, in particular Kv4.3 and Kv4.2 channels are downregulated increasing action potential duration and arrhythmogenesis. During pregnancy, there is an increased risk of arrhythmias, the heart develops functional hypertrophy, and hormone levels dramatically change; however, no studies are available on changes in K+ expression/function induced by sex hormones, that may explain the cardiac risks during pregnancy or their reversibility after delivery. Thus, the main hypothesis is that, during pregnancy and postpartum, sex hormones may regulate I to current components (e.g. Kvl.4, Kv4.2, Kv4.3, KChlP2, MiRP1, frequenin) in a genomic or non-genomic fashion. Preliminary Studies show that: (a) cardiac Kv4.3, Kvl.4 and KCh/P2, but not Kv4.2 channel transcripts, and Kv4.3 protein were reduced in late pregnancy; (b) as in pregnancy, 17-beta-estradiol (E2) treatment reduced cardiac Kv4.3 and Kvl.4 mRNA; (c) E2 reduced Kv4.3 protein expression in cultured adult myocytes; (d) E2 had dual effects on I to currents, at 100 nM (similar to late human pregnancy), it shortened the action potential and increased I to amplitude; whereas, an opposite effect was produced by 10 fM E2; (e) c-Src tyrosine kinase, which is activated by E2 and from the onset of hypertrophy, reduced expression of Kv4.3; and (f) tyrosine kinase activation produced action potential prolongation and I to current reduction. We will mainly use mice and multiple experimental approaches. The Specific Aims are to: (1) Investigate, during pregnancy and early postpartum, the remodeling of action potentials, and of I to fast (I to-f) and I to slow (I to-s) currents, and underlying molecular components. (2) Investigate the action of E2 on I to-f/I to-s currents and their molecular components, and if E2 stimulates Kv4.3 gene transcription through the c-Src/MAPK (ERK) axis. (3) Characterize and define the mechanism(s) of non-genomic regulation of I to-f and Kv4.3 channels by E2. (4) Investigate short- and long-term actions of c-Src-dependent tyrosine phosphorylation on I to currents and Kv4 isoforms. (5) Determine the motifs in Kv4.3 involved in its regulation by c-Src. These studies should provide new information on the cellular and molecular mechanisms leading to the remodeling of cardiac K+ channels in the early stages of hypertrophy, and help in the design of new strategies for preventive Medicine.

DESCRIPTION (provided by applicant): Potassium channels are key regulators of smooth muscle contractile state. Accumulating evidence suggests that they may act as signal transducers, translating extracellular stimuli by hormones, neurotransmitters and peptides, to intracellular signaling processes. To undertake this task, K+ channels must be in close proximity and forming macromolecular complexes with membrane receptors and intracellular signaling molecules. Moreover, this association should be dynamic and susceptible to change under different physiological conditions, for example, under the influence of sex hormones during pregnancy. Thus, our main hypothesis states that K+ channels localize to specific microdomains in smooth muscle (SM) and are intimately associated with receptors and signaling cascades either directly or indirectly via scaffolding proteins, and that, this association and subcellular distribution is influenced by sex hormones. To test this hypothesis, we will use as model system uterine SM that undergoes dramatic remodeling in structure and function during pregnancy and postpartum, and a multidisciplinary approach analyzing functional association, native subcellular colocalization, molecularity of protein-protein interactions, and the hormonal mechanisms leading to remodeling. Studies will focus on voltage and Ca2+-activated K+ (MaxiK, BKca) and Kv4.3 channels, 5-HT receptors, c-Src tyrosine kinase and caveolins, all critical regulators of SM function. Preliminary data indicate that, in myometrium: i) a new c-Src-related protein with tyrosine kinase activity is further induced in late pregnancy, ii) c-Src, Kv4.3 and caveolin comigrate in detergent resistant fractions and can be coimmunoprecipitated, iii) recombinant MaxiK carboxyl tail interacts with c-Src, iv) MaxiK/Kv4.3 in single myocytes are clustered mimicking c-Src, v) c-Stc and caveolin-la mRNAs are quadrupled during pregnancy, vi) spontaneous contractility is under the control of Src kinase <-> tyrosine phosphatase, and vii) its mechanical output is regulated by the new agonist ->c-Src tyrosine kinase ->MaxiK pathway. Thus, the Specific Aims are to investigate: 1) the identity of a new pregnancy-induced Src-like protein; 2) whether in SM MaxiK/Kv4.3 form macromolecular complexes with c-Src, caveolin and 5-HT receptors, their subcellular colocalization, and potential remodeling across gestation; 3) the molecular interactions of MaxiK-c-Src-caveolin-receptor comp/exes; 4) the mechanism(s) triggered by sex hormones leading to pregnancy-related changes of c-Src-caveolins- MaxiK/Kv4.3 associations, and their recovery in postpartum; and 5) the functional impact and mechanism of Src modulation of MaxiK/Kv4.3 channel activity, and their role in spontaneous or 5-HT induced contractility. These studies will increase our understanding of SM biology and may lead to better treatments of SM-related pathologies.

DESCRIPTION (provided by the applicant): To better understand cell function in health and disease, we need to visualize the localization of protein complexes and dynamic changes in different cellular compartments in response to normal stimuli or insult. To this end, we will develop "Nanomicroscopes" for fluorescence imaging to measure structures and their dynamics inside a cell with a 3D spatial resolution down to the scale of 20-40 nm while maintaining the microscopic whole cell scale over a 20-100 um range. In a multi-disciplinary engineering and biological sciences effort, we will develop and apply such "Nanomicroscopes" to cardiovascular research, specifically to a pressure-overload model of heart failure. The overall hypothesis states that, stress-induced structural rearrangements -in the subcellular location and interactions- of key signaling protein complexes in the heart and blood vessels differentially contribute to the onset and progression of heart failure. We show exciting preliminary advances in the design of a novel Reflexion Nanomicroscope that achieves a full-width-half- maximum (FWHM) of ~100 nm lateral resolution. The Specific Aims are: Aim 1. TO DEVELOP NOVEL NANOMICROSCOPIES TO MEASURE STATIC AND DYNAMIC PROTEIN-PROTEIN INTERACTIONS. 1.1. To further improve the novel Reflexion Confocal Nanomicroscope by constructing a fast acquisition multicolor Reflexion Confocal with FRET for living cells and develop the theory to enhance its resolution beyond. 1.2. To combine STED with 4Pi microscopy to achieve 10-20 nm 3D resolution and expand to two fluorescene wavelengths for protein colocalization imaging. Aim 2. TO APPLY THE NOVEL NANOMICROSCOPES TO VISUALIZE STATIC AND DYNAMIC CHANGES OF MACROMOLECULAR COMPLEXES REGULATING HEART AND VASCULAR SIGNALING IN A PRESSURE OVERLOAD MODEL OF HEART FAILURE BY DETERMINING: 2.1. The structural basis of local stress signaling (p38 kinase signalsome) and EC-coupling defects in cardiomyocytes under stress and heart failure. 2.2. The spatiotemporal remodeling of proteasome subunits and their assembly in normal, stressed and protected (e.g. estrogen signals) myocardium. 2.3. The stress-induced dynamics/remodeling of aortic GPCR-Src tyrosine kinase signaling complexes that exacerbate heart failure. Nano-imaging will be complemented by state-of-the-art molecular manipulations, biochemical and proteomic approaches. These studies will be the basis to unravel -at the nanoscale level- the structural map of protein complexes at the subcellular level, their localization and dynamic interactions in cardiovascular disease. Identifying the structural basis of cell signaling pathways/networks will provide opportunities to discover new therapeutic targets to alleviate cardiovascular disease, a leading cause of death in the United States.

DESCRIPTION (provided by applicant): Our long term goal is to obtain an integral view of the mechanisms by which Thromboxane A2-prostanoid receptor (TPR) and the large conductance Ca2+-activated K+ channel (MaxiK, BK) interact with each other to regulate vascular function. TPR and MaxiK play significant roles in determining vascular health. In addition, both proteins are known to be involved in the modulation of tumorigenesis and myocardial infarction. In coronary arteries, TPR are activated by the prostanoid thromboxane A2 (TXA2) leading to powerful vasoconstrictions;while MaxiK channel aided by its b1 subunit can fine tune arterial tone determined by the degree of channel activity. Our early work showed that TXA2 mimetic U46119 inhibits MaxiK channel activity in membranes from coronary smooth muscle reconstituted in lipid bilayers, which suggested a strong functional association between both TPR and MaxiK that endured dissociative reconstitution procedures. Recent preliminary experiments also show that: 1) TPR modulates MaxiK pore-forming a subunit (Slo1) in a dual way via novel mechanisms: i. constitutive activation (as a regulatory b subunit?), and ii. agonist-induced inhibition in a G-protein independent manner, where MaxiK channel activity can be reduced by the specific TPR agonist U46619, 2) TPR and MaxiK channel subunits form heteromultimeric complexes in native arteries and in expression systems, 3) TPR and MaxiK channel functional coupling occurs in native human coronary arterial myocytes and can be reproduced after ectopic expression of TPR and Slo1, and 4) agonist-stimulation enhances TPR and Slo1 association. Here, we will test the new hypothesis that the TPR can act as a positive regulatory subunit of Slo1 in a constitutive manner and that agonist stimulation switches the TPR-induced modulatory action on the channel from activatory to inhibitory. Parallel experiments in model and native systems will link molecular mechanisms to functional consequences resulting from the tripartite interaction among TPR, Slo1 and its b1 subunit. The specific aims are to: 1. Determine the mechanism(s) underlying constitutive TPR positive modulation of Slo1 channel activity, 2. Investigate the role of b1 in and physiological consequences of TPR-Slo1 constitutive activation, 3. Unravel the molecular mechanism(s) of Slo1 channel inhibition by agonist (U46619)- activated TPR, and 4. Define the role and functional consequences of b1 subunit in agonist-TPR-Slo1 channel inhibition. Experiments will be performed using modern approaches such as biochemistry, molecular biology, and opto-biophysical methods including fluorescence microscopy at the diffraction limit. Because of the broad impact of MaxiK and TPR in mammalian physiology, the knowledge derived from these studies may provide new opportunities for the prevention of disease not only in the cardiovascular system but in other systems as well. PUBLIC HEALTH RELEVANCE: Thromboxane A2-prostanoid receptors (TPR) and the large conductance Ca2+-activated K+ channel (MaxiK, BK) are proteins involved in the regulation of vascular tone and have been implicated in the development of (i.e. TPR) or protection against (i.e. MaxiK) cardiovascular diseases like hypertension, heart attack and stroke. Our discovery that the 1 (Slo1) and b1 subunits of MaxiK interact with TPR and the studies proposed here will set the basis to understand in detail vasoconstricting mechanisms that afflict cardiovascular function, which is crucial to ultimately design new therapeutics targeting cardiovascular disease.

DESCRIPTION (provided by applicant): The large-conductance, Ca2+-activated K+ channel from cardiac mitochondria (mitoBKCa) is thought to play a role in cardioprotection. MitoBKCa molecular size is uncertain with reported immunochemical signals at ~55 and ~125 kDa. In addition, mitoBKCa molecular identity and its mitochondrial targeting mechanisms remain unknown, while there is scarce information about its functional properties or direct evidence for their role in cardioprotection. Because cardiac mitoBKCa shares conductance, Ca2+ responsiveness, and sensitivity to pharmacological agents with its plasma membrane counterpart known as BKCa (or MaxiK), we expect that mitoBKCa is assembled like BKCa by four pore-forming a subunits with a monomeric mass of ~125 kDa. We will now test the hypotheses that: 1) mitoBKCa and plasma membrane BKCa are encoded by the same gene and splice variation provides BKCa with intrinsic signals for its preferential mitochondrial targeting; 2) the normal absence of BKCa from the cardiomyocyte plasmalemma and presence in mitochondria is ruled by both an intrinsic signal(s) within mitoBKCa backbone (i.e. splice insert) either directly or indirectly (i.e. via a chaperone), and by cell-specific mechanisms, and ) mitoBKCa contributes to cardioprotection by regulating mitochondrial calcium retention capacity (CRC) and permeability transition pore (mPTP) opening. Preliminary Data shows: 1) the detection of a ~125 kDa protein in mitochondria by specific anti-BKCa antibodies; 2) the detection of all 27 constitutive BKCa exons in isolated cardiomyocyte mRNAs; 3) that BKCa isoform containing splice insert DEC (C-terminal insert of 61 amino acids) but not the constitutive form of BKCa (insertless BKCa) is readily targeted to mitochondria in adult cardiomyocytes; 4) that mitoBKCa subproteome uncovered as a partner Hsp60, a heat shock protein relevant for folding of mitochondrial imported proteins; and 5) that BKCa gene ablation prevents the cardioprotective action of putative BKCa channel opener NS1619. Overall the data support the above hypotheses, which will be tested using multiple approaches and pursuing the following Specific Aims to: 1. Identify the molecular correlate of cardiac mitoBKCa; 2. Functionally validate the identity of cloned putative mitoBKCa; 3. Determine signal mechanisms involved in mitoBKCa mitochondrial targeting; and 4. Directly address the role of mitoBKCa in cardioprotection. The outcomes of this program will open the opportunity to study mitoBKCa at the molecular level and advance the cardiac field by: solving mitoBKCa identity, providing information on its targeting mechanisms, and defining its functional properties and role in cardioprotection. Further understanding of the underlying molecular mechanism(s) of mitoBKCa cardioprotective effects will provide new targets for translation into therapeutics. PUBLIC HEALTH RELEVANCE: One of the mechanisms involved in protecting the heart from lack of oxygen like that occurring during heart infarct is thought to be the opening of a mitochondria potassium channel named mitoBKCa. Here, we propose to unveil mitoBKCa molecular identity, the mechanisms that target it to mitochondria and directly demonstrate its role in protecting the heart from injury by oxygen deprivation. The results of this investigation wll allow the advancement of cardioprotective medicine and provide new molecular targets for therapeutics.