Michael M. Tamkun - US grants

The sodium/potassium-ATPase, or Na+ pump, is the protein responsible for the ATP dependent transport of sodium and potassium ions across cell membranes. While all cells of the body contain this essential enzyme, it is especially important in nerve and muscle because the electrical properties of the membrane are dependent on these two ion gradients. The transmembrane Na+ gradient maintained by the ATPase also provides the energy for numerous membrane co-transport mechanisms. The cardiac ATPase is the generally accepted receptor for digitalis, a drug commonly used in the treatment of congestive heart failure. Recent data indicate that a family of genes is responsible for different isoforms of the ATPase alpha subunit in species as diverse as chickens, rodents, and man. Only one isoform of the beta subunit has been identified in any species. The alpha subunit genes show tissue-specific expression, suggesting that the various ATPase isoforms have evolved to satisfy the ion transport needs of specific cells. The biological significance of ATPase isoforms will be examined by comparing functional differences between isoforms and by localizing the various isoforms to specific cells within complex tissues. Mouse L-cells will be transfected with chicken ATPase cDNA and the expression of the chicken subunits monitored by species-specific fluorescent antibody binding. Differences in ouabain sensitivity between the endogenous and expressed ATPase will allow pharmacological separation of mouse and chicken ATPase function. This expression system will be used to quantitate the ouabain sensitivity, ion transport activity, and ATPase activity of the expressed chicken isoforms. Co-expression of the alpha subunit isoforms with and without the beta subunit may identify an effect of the beta subunit on alpha subunit function. Isoform localization will be approached using two methods. In situ hybridization with isoform-specific cRNA probes will localize isoform mRNA to specific cells within brain, muscle, heart, and kidney. Isoform-specific antibodies will be produced against synthetic peptides and immunofluorescence techniques used to examine cell specific isoform expression. These site-directed antibodies will also be valuable reagents with which to study structure function relationships in future experiments. Correlation of ATPase isoform function with cellular localization will greatly increase our knowledge of ion transport physiology. Correlation of isoform function with the amino acid sequence substitutions that define each isoform will improve our understanding of the structure/function relationships involved in ion transport.

DESCRIPTION (adapted from the applicant's abstract): Voltage-gated K+ channels, responsible for action potential repolarization and setting the resting membrane potential, are proteins encoded by one of the most complex group of ion channel genes found in the cardiovascular system. A small change in K+ permeability in vascular smooth muscle membranes, results in a significant change in membrane potential and in Ca2+ channel activity. Thus, changes in K+ channel function can have an important effect on vascular tone. Three specific aims will be examined in the proposed work. 1. K+ channel subunit localization and assembly will be analyzed in rat cardiac myocytes and vascular smooth muscle tissue. Two hypotheses will be examined: (i) In heart Kva1.5 is either a homomeric alpha structure or assembled with Kva1.2 at the intercalated disk. In the ventricle (but not the atrium) the Kva1.5-containing complex is assembled with an inactivation-conferring Kvb subunit. (ii) In vascular smooth muscle, Kva1.5 exists as a heteromeric structure in association with the Kva1.4 subunit and the Kvb1.2 beta subunit. 2. The mechanisms underlying potassium channel a/b subunit interactions will be determined. Two hypotheses will be examined. (i) ab assembly involves the association of nascent channels with chaperon-like proteins that facilitate subunit assembly. (ii). Specific amino acids on b subunits determine physical ab interactions. The beta effects on voltage-sensitivity, deactivation, and inactivation can be traced to different amino acids. Furthermore all ab interactions occur at the N-terminal domains of both the alpha and beta subunits. 3. The cellular and subcellular distribution of the IKr potassium channel protein, h-erg, will be determined in normal and diseased human myocardium. One hypothesis will be investigated: IKr expression is not static, with localization being altered in diseased myocardium. The immunohistochemistry in aims 1 and 3 will be performed primarily on rat, canine and human tissue sections with antibodies that currently exist and antisera under production. Alpha-beta interactions will be monitored functionally by voltage-clamp techniques and physically by immunopurification. Subunit assembly will be determined by immunopurification methods where purification of two distinct subunits with an antibody specific for only one is the operational definition of assembly. The amino acids involved in this interaction will be identified by a variety of mutagenesis approaches.

DESCRIPTION: Ion channel regulation within neuronal and muscle membranes is an important determinant of electrical excitability in the nervous and cardiovascular systems, skeletal muscle, GI tract, and uterus. Voltage-gated K+ channels (Kv channels) play an important role in setting the resting potential and determining repolarization in these functionally diverse systems. Recent evidence suggests that specialized microdomains commonly referred to as lipid rafts exist within the plane of most plasma membranes. These domains are enriched in cholesterol and sphingolipids and concentrate a number of signal transduction molecules. In the Preliminary Data section, we demonstrate that the voltage-gated K+ channels, Kv2.1, Kv1.1, Kv1.5, and Kv1.4, but not Kv4.2, target to lipid rafts in both heterologous expression systems and rat brain. In addition, Kv2.1 and Kv1.5 probably reside in different raft compartments. Depletion of cellular cholesterol alters the buoyancy of the Kv2.1-associated rafts and shifts the midpoint of Kv2.1 inactivation by 30-40 mV without affecting peak current density or channel activation. Incubation of Kv2.1 expressing cells with fumonisin B, an inhibitor of ceramide synthase, causes a similar shift in the inactivation curve. Ceramide is both a raft component and an intracellular signaling molecule. Thus, raft association is functionally significant, for such a shift in the inactivation will result in a large percentage of the Kv2.1 channels being functionally silenced in the range of physiological membrane potentials. In addition, the preliminary data suggest fumonisin B induces mistargeting of Kv2.1 from the cell body to the distal dendrites in cultured neurons, suggesting raft-related signaling mechanisms are involved in Kv2.1 targeting. The Specific Aims will 1) address the mechanisms involved in the targeting of Kv2.1 to lipid rafts, with emphasis placed on subunit composition and channel transmembrane domains; 2) examine the functional significance of Kv2.1 association with lipid raft domains with emphasis on ceramide signaling pathways, 3) examine the relationship between raft association, ceramide signaling, and cell surface localization in neurons; and 4) begin initial work aimed at purification of Kv 2.1-containing lipid rafts from brain. This proposed research examines a new area in Kv channel research that will have important implications in multiple tissue systems.

DESCRIPTION (provided by applicant): Tremendous advances in ion channel research have been made with respect to channel structure and function. However, there is a lack of information regarding how living cells process ion channel proteins in real time. Such information is important since rapid modulation of ion channel surface expression is likely to represent a central mechanism in the regulation of cellular electrical excitability. Human diseases have already been linked to ion channel localization and trafficking defects. The Kv2.1 delayed-rectifier K+ channel targets to unique cell surface clusters in hippocampal neurons. Since these structures are regulated in vivo by stimuli associated with neuronal injury, i.e. hypoxia, ischemia, and excess neurotransmitter release, these microdomains are likely to participate in the neuro-protective response during such insults. However, Kv2.1 also plays a role in apoptosis, with this Kv isoform providing the increased K+ permeability required for the completion of the apoptotic program in cortical neurons. These diverse functions mandate that Kv2.1 activity and trafficking be tightly regulated. Our overall hypothesis is that the Kv2.1 surface clusters are central in the regulation of both Kv2.1 trafficking and function. Thus, a better understanding of cluster function will improve our ability to manage stroke-related issues. By clustering these channels within a defined surface compartment that serves as a platform for both insertion and retrieval, the regulation of trafficking is more efficient than if the channel is homogenously distributed over the cell surface. In addition, our preliminary data suggest that channels within the surface clusters do not conduct K+ until released from these cell surface domains, suggesting that K+ current density is controlled by more than transport to the cell surface. The proposed research will employ a multidisciplinary approach utilizing live cell imaging, single channel detection/tracking, site-directed mutagenesis and electrophysiology. The three Specific Aims will test the hypotheses that 1) Kv2.1 clusters represent cell surface platforms for channel insertion and retrieval, 2) stimulus-induced modification of the cortical cytoskeleton causes immediate release from the cell surface cluster and 3) Kv2.1 surface clusters are cell surface storage sites containing non-conducting channel. This research will have implications far beyond neurobiology, for Kv2.1 is the most widely expressed Kv channel and is also physiologically important to the cardiovascular and endocrine systems. PUBLIC HEALTH RELEVANCE: The proposed research examines the localization of the Kv2.1 potassium channel in brain neurons. This localization is essential in the neuro-protective response to stroke injury and hyperactivity in the brain. Mice lacking the Kv2.1 channel are epileptic and hyper-excitable. Understanding the molecular mechanisms underlying Kv2.1 localization will enhance the treatment of stroke and epilepsy.

In this project the PIs will study the regulation of Kv2.1 channel clusters that may play several important biological roles in the brain. Currently, the mechanism by which clusters Kv2.1 are regulated and maintained is unknown. The project focuses on the biophysics of Kv2.1 voltage-gated K+ channel cell surface dynamics with particular emphasis on cytoskeleton-membrane interactions in live cells. The overall goal of the research is to improve our understanding of the mechanism by which the cortical cytoskeleton functionally forms a diffusion limiting fence that selectively corrals a sub-population of Kv2.1 channels. The specific research aims are: (1) characterize the dynamics of clustered and non-clustered channels within various surface regions of cultured neurons, (2) measure the influence of the cortical cytoskeleton and raft microdomains on Kv2.1 channel dynamics, (3) build a microscope to implement high-speed particle tracking and optical tweezers, and (4) determine the mechanism that forms Kv2.1 clusters on the cell surface. This experimental work will lead to unique insights in the molecular mechanism of membrane protein dynamics. The project focuses on investigating Kv2.1 clustering regulation, dynamics and interactions with the cytoskeleton which may lead to improved treatments for acute ischemic stroke through enhanced neuro-protective approaches. The research program will be integrated with an educational component by building an optical tweezers setup for educational purposes. Under-represented students will be recruited at minority meetings and through a Women & Minorities in Engineering Program at Colorado State University.

The project is jointly sponsored by the Physics and the Molecular Cell Biology Divisions at NSF.

DESCRIPTION (provided by applicant): Voltage-gated Na+ (Nav) channels initiate the majority of action potentials in the nervous system and alpha subunit mutations are responsible for pathologies ranging from an absence of pain phenotype to epilepsy. A high Nav channel density at the axon initial segment (AIS) and node of Ranvier is believed to regulate action potential threshold within these neuronal compartments. Mechanisms regulating the targeting to these sub- cellular domains, and the functional consequences of such localization, are directly relevant to human health since diseases such as epilepsy are caused by Na+ channel trafficking or localization defects. In addition, loss of appropriate Na+ channel localization to te axon initial segment after ischemic injury likely contributes to neuronal dysfunction and modulation of this relocalization could be a future treatment for stroke. Febrile seizures may originate in the AIS since increased temperature enhances the activity of AIS localized Nav channels. Interestingly, the AIS is structurally remodeled following traumatic brain injury and in models of central nervous system trauma Nav channel blockers are neuro-protective. Despite the physiological and pathological significance of the Nav channels in the AIS, little information exists concerning the trafficking, maintenance, and location-dependent function of these channels. In fact, major debates in the field center around questions such as how Nav channels traffic to the AIS and what percentage of these channels are functional. Research in these areas has been hampered by a lack of fluorescently tagged Nav channel constructs and live cell imaging approaches. This research proposal utilizes novel Nav channel constructs in conjunction with high resolution single molecule imaging approaches to monitor the real-time trafficking, localization, and function of Nav1.6 channels in the soma and AIS of hippocampal neurons. Specific Aim 1 will test hypotheses relating to the mechanisms of Nav1.6 localization at the axon initial segment. Specific Aim 2 will examine how Nav1.6 channel activity varies as a function of cell surface location. Hypotheses common to both aims deal with how Nav1.6 responds to ischemia-like neuronal insults. The proposed research will enhance our understanding of the function and regulation of neuronal Nav channels. Preventing the altered Nav channel localization induced by ischemic insult is a potential target for future drug development.

DESCRIPTION (provided by applicant): Endoplasmic reticulum/plasma membrane (ER/PM) junctions are best understood in muscle and immune cells where they mediate contraction and lymphocyte activation. Despite the early electron microscopic detection of sub-surface cisterns in neurons the function and molecular interactions responsible for the maintenance of this membrane junction are poorly understood. Our preliminary data demonstrate that the Kv2.1 delayed rectifier K+ channel plays a central role in the formation of neuronal ER/PM junctions. This channel forms highly stable cell surface clusters on the neuronal soma that reside in close apposition the ER membrane. Most of these localized channels are non-conducting and play a direct structural role by enhancing the ER/PM junctions and dramatically increasing the junction surface area, likely by binding unknown ER membrane proteins. Our published data indicate that the Kv2.1 enhanced ER/PM junctions are trafficking hubs, providing platforms for delivery and retrieval for multiple types of membrane proteins. In addition, our preliminary data indicate that calcium signaling proteins localize to the neuronal Kv2.1/ER/PM junction. We propose the Kv2.1-stabilized ER/PM junctions represent a macromolecular plasma membrane complex that functions as a scaffolding site for both membrane trafficking and Ca2+ signaling. Given that this complex is regulated by stroke-related neuronal insults, an improved understanding of the components, function and dynamics within this cell surface microdomain is needed. This proposal assembles an interdisciplinary team from four institutions with combined expertise in intracellular Ca2+ dynamics, ion channel electrophysiology, molecular and cell biology, nanoSIMS cellular imaging technology, high-resolution real time imaging, optics, and quantitative analysis of single molecule diffusion. Aim 1 seeks to identify the proteins and lipids involved in the Kv2.1/ER/PM complex, Aim 2 examines L-type calcium channel function at the Kv2.1/ER/PM junction and Aim 3 studies calcium channel/beta2 adrenergic receptor dynamics within this domain.

Endoplasmic reticulum/plasma membrane (ER/PM) junctions are known to be sites of calcium ion (Ca2+) influx. Recently, the PI discovered that these junctions function as trafficking hubs for insertion and removal of plasma membrane proteins. Furthermore, the PI has found that the voltage gated potassium channel Kv2.1 interacts with the endoplasmic reticulum, dramatically increasing ER/PM junction surface area and structurally changing the junction morphology. The PI's findings show that the Kv2.1 potassium channel remodels to cortical ER, which is likely within 30 nm of the plasma membrane. Kv2.1 is playing a structural role similar to that of Orai, for the PI proposes that Kv2.1 is binding an ER membrane protein. Thus, Kv2.1-mediated ER enrichment on the cell surface is a novel specialized organelle with specific functions in protein transport vital to cell signaling.

The current project focuses on understanding the biology of ER/PM junctions with particular emphasis on the regulation of the ER/PM junction structure and its function in the modulation of membrane protein trafficking. The PI will answer the following questions: What is the role of Kv2.1 in protein trafficking at ER/PM junctions? How are ER/PM junctions dynamically regulated by Kv2.1? What are the relationships between the cortical cytoskeleton, ER, and Kv2.1? Which theoretical framework can be used to describe the assembly and maintenance of these domains? How does large-scale membrane behavior emerge from the interactions between Kv2.1 and ER? The fusion of multicolor single-molecule tracking in living cells and advanced stochastic process analysis, which are integral to the project, will provide answer to these questions. This research will offer excellent opportunities for graduate and undergraduate student participation in interdisciplinary research through the collaboration between two laboratories with very different backgrounds. The research program will be integrated with an outreach component by developing a microscopy laboratory for students at a local elementary school. The goal of the outreach program is to foster scientific enquiry and to motivate students to appreciate science from an early age. This lab presents a unique opportunity to leverage integration of education and research, giving students access to hands-on practical learning.

This project is being jointly supported by the Physics of Living Systems program in the Division of Physics and the Cellular Dynamics and Function Program in the Division of Molecular and Cellular Biosciences.

The Kv2.1 K+ channel is the most abundantly expressed and widely distributed voltage-gated K+ channel in mammals. Our previous research demonstrates that in addition to functioning as a delayed rectifier K+ channel and regulating plasma membrane potential, a non-conducting, majority population of Kv2.1 forms endoplasmic reticulum/plasma membrane (ER/PM) contact sites. In hippocampal neurons Kv2.1 channel binding to the cortical endoplasmic reticulum generates micron-sized Kv2.1 clusters on the surface of the soma, proximal dendrites and axon initial segment. Data in the literature indicate that ER/PM junctions regulate neuronal burst firing, the non-vesicular lipid transfer directly from the ER to the cell surface, and plasma membrane PIP2 levels. Our preliminary data show that the Kv2.1-induced ER/PM junctions, but not other ER/PM junctions, alter ER Ca2+ homeostasis, plasma membrane organization, and exocytosis. Interestingly, Kv2.1 interaction with the cortical ER is regulated by neuronal activity and stroke-like insults such as hypoxia, ischemia and excess glutamate, indicating that the functions linked to these microdomains are remodeled following hyperactivity or neuronal insult. Thus, the proposed research examines a novel non-conducting function of Kv2.1 that 1) is central to neuronal physiology and 2) is regulated by neuronal activity, insult and stroke. The three Specific Aims will address the molecular mechanisms by which Kv2.1 alters ER Ca2+ homeostasis and membrane protein localization at somatic ER/PM junctions and exocytosis at presynaptic ER/PM contacts. Aim 1. To test the hypothesis that Kv2.1-induced ER/PM contact sites enhance store-operated Ca2+ entry by providing localized K+ conductance. Preliminary data suggest that ER Ca2+ refilling is enhanced in neurons expressing Kv2.1. Aim 2. To test the hypothesis that the concerted action of Kv2.1 and cortical actin controls the localization of Ca2+ signaling proteins in the vicinity of ER/PM junctions. Preliminary data indicate Kv2.1-induced ER/PM junctions influence the cell surface distribution of Cav1.2, BK K+ channels and b2 adrenergic receptors. Aim 3. To test the hypothesis that synaptic vesicle exocytosis is modulated by Kv2.1 channels at the ER/PM junction in presynaptic terminals. Preliminary data demonstrate that both endogenous and transfected Kv2.1 is localized at presynaptic terminals and that shRNA-based knockdown of Kv2.1 suppresses glutamatergic vesicle exocytosis by 50% without affecting the action potential. While Kv2.1 point mutations that cause human epileptic encephalopathy alter channel conductance, a subset of point mutants that are linked to developmental delay induce premature stop codons in the channel C-terminus that should not affect conductance. Instead, these mutations are predicted to only prevent Kv2.1 binding to the cortical ER. Thus, mutations affecting both the conductance and cortical ER remodeling roles of Kv2.1 underlie human disease. The research in this proposal will substantially advance our understanding of the role that Kv2.1- containing ER/PM contact sites play in neuronal physiology.