John H. Caldwell - US grants

This research will focus on the distribution of ion channels in skeletal muscle and the mechanisms for creating and maintaining these channel distributions. The distribution and single channel properties of voltage-gated sodium channels will be studied with loose patch and tight patch voltage clamp. A new technique which combines both patch voltage clamp and ionophoresis will be used to find the degree of colocalization of sodium channels and acetylcholine receptors. Different distributions of sodium channels in fast and slow muscle may suggest functional roles for these channel distributions. Potassium channel and choride channel distributions will be studied with the same techniques since there is evidence that these too are nonuniformly distributed in muscle. The pertinence of extending these studies to other channels is that the channels are not regulated in unison either spatially or in response to perturbations. Thus it may be possible to identify both general and specific regulatory controls. Extracellular factors that may regulate channel distribution or immobilization will be tested.

A multidisciplinary approach will be used to determine the distribution of ion channels in skeletal muscle ad to study mechanisms for creating, controlling, and maintaining these channel distributions. The density of channels along the length of the cell will be determined with new electrophysiological methods. The site of synthesis of the channels will be determined with in situ hybridization and immunocytochemistry. The enhanced spatial resolution of these electrical and histological techniques is revealing a complex ordering of ion channels. Ion channels are particularly important regulators of excitability in neurons and skeletal muscle. How the control of synthesis and localization of channels and other membrane proteins is accomplished is a fundamental question for all cells. The distribution and single channel properties of voltage-gated sodium channels will be studied with loose patch and tight patch voltage clamp. A new technique which combines both patch voltage clamp and ionophoresis will be used to find the degree of colocalization of sodium channels and acetylcholine receptors. Different distributions of sodium channels in fast and slow muscle may suggest functional roles for these channel distributions. Potassium channel and chloride channel distributions will be studied with the same techniques since there is evidence that these too are nonuniformly distributed in muscle. The pertinence of extending these studies to other channels is that the channels are not regulated in unison either spatially or in response to perturbations. thus it may be possible to identify both general and specific regulatory controls. The control of channel distribution could occur at any of a number of steps. Since skeletal muscle cells are multinucleated, it is possible that nuclei within the same cytoplasm are differentially regulated. This will be studied with in situ hybridization to determine the abundance and location of mRNA for the sodium channel and acetylcholine receptor. Labeled antibodies and snake toxin will be used to follow protein synthesis of these channels. Extracellular and intracellular factors that may regulate channel synthesis, distribution, or immobilization will be tested. These studies will also be done another multinucleated cell, the eel electrocyte, which also segregates membrane proteins (sodium channels, acetylcholine receptors, and sodium-potassium pumps).

Voltage-gated sodium channels (NAChs) are vital for electrical signaling and conduction in the nervous system. There are at least ten NaCh genes in rodents, and each gene has a known ortholog in humans. The specific roles of each NaCh subtype are unknown. One hypothesis is that subtypes are differentially distributed and modulated. This proposal is focused upon the molecular basis for modulation and localization of brain NaCh subtypes. The goal of Aim 1 is to identify proteins that bind to the cytoplasmic domains of brain NaChs (esp. Nav1.6) and are responsible for (a) modulation of channel fiinction and (b) targeting to different subcellular sites. Two complementary methods are being used to find and isolate proteins associated with NaChs: (1) the yeast two-hybrid assay and (2) protein purification/mass spectrometry. Aim 2 is focused upon the interactions of NaChs with calmodulin, which was isolated with the yeast two-hybrid assay. This interaction with brain NaChs will be characterized biochemically and electrophysiologically. Aim 3 utilizes imaging techniques to study the subcellular distribution of brain sodium channels (esp., Nav 1.6) and their binding proteins. After characterizing this distribution, the effects of mutations in NaChs or in the binding proteins identified in Aim I will be studied. This research has both basic science and clinical relevance. Ion channels exist in complexes with other membrane, extracellular, and intracellular proteins. To understand the behavior of these channels, it is important to know which proteins are present in these complexes and hew the proteins interact with the channel. Mutations in muscle NaChs are responsible for some disorders of skeletal muscle and for long QT syndrome in cardiac muscle. It is expected that mutations in brain sodium channels and in the proteins that bind to NaChs will produce CNS disorders in humans, and the proposed studies will contribute to our understanding of these disorders.

The Golgi complex is present in every eukaryotic cell, from yeast to humans, and functions in posttranslational protein modifications and sorting of these molecules to post-Golgi destinations. Both processes require an acidic lumenal pH and transport of substrate and reactants into and out of the Golgi lumen. Endogenous ion channels are expected to be important for regulating the ionic environment within the Golgi lumen. This proposal is to isolate and characterize these endogenous ion channels of the Golgi complex and is a collaboration between two laboratories, one that studies ion channels and one that studies Golgi function. Single ion channel studies are now feasible because we have recently improved the isolation of a Golgi fraction from rat liver. We eliminated proteins transiting the Golgi and achieved a 400-700 fold enrichment of endogenous Golgi proteins. Ion channels in the enriched fraction have been incorporated into planar lipid bilayers. We named the most prevalent ion channel GOLAC1 (Golgi Anion Channel 1). This channel has novel properties and is modulated by pH on the lumenal surface. Two hypotheses are proposed for the function of the GOLAC 1: first, it provides counterions necessary for acidification of the Golgi lumen by an electrogenic H+ATPase and second, it removes phosphate (generated by glycosylation and sulfation) from the Golgi lumen. There are three specific aims. (1) Electrophysiologically characterize anion and cation channels of the Golgi. (2) Obtain peptide and cDNA sequence by enriching for channel activity with subfractionation of detergent- solubilized Golgi proteins. Proteins that enrich in parallel with channel activity will be identified using 2D-gel electrophoresis, mass spectrometry, and peptide sequencing. The cDNA identified will be expressed, purified, and confirmed to be a GOLAC in bilayer studies and a Golgi protein by immunofluorescence. (3) Study modulation of Golgi channels by candidate molecules and cell factors. From a clinical viewpoint, there are an increasing number of diseases classified as ion channelopathies. It is likely that some human diseases will be due to mutations of endogenous Golgi channels.

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An Optical Probe capable of Activating/Reporting on axon activity in nerves of parasympathetic nervous system would be a boon to researchers working with the pancreas. We are proposing to develop such a Probe using our background and experience in optogenetics in the peripheral nerve, bio-imaging and compact multiphoton microscope design. Current neuro-modulation approaches for the vagus nerve are generally all or nothing events that cause simultaneous changes in heart rate, for example, along with changes in pancreatic function. We propose to develop a novel compact Optogenetic based Optical Probe capable of optically neuromodulating individual afferent and/or efferent axons within nerves of the parasympathetic, or peripheral, nervous system. We seek to read-in or read-out from these nerves with the goal of modulating organs or brain circuits innervated by them. Our central premise is that we can use optics to communicate with axons in a nerve. For optical approaches to work we need to convert action potentials into an optical signal. This can be done using reporter proteins or by some other means that is ancillary to action potential generation. Because nerves do not naturally express optical proteins, we will work with transgenic mice that express these proteins and use these mice to refine our system before making it available for other researchers to use. We will develop a bench-top Optical instrument that can be shared with other research teams to allow us, and them, to interrogate specific fascicles and axons within mouse, and ultimately human, nerves. Our goal here is the vagus nerve and its innervation of the pancreas. The vagus nerve is one of the main conduits into the parasympathetic nervous system. The ability to interface with this nerve gives one the ability to neuromodulate the viscera in one direction and the brain in the other. We are proposing to couple an optical fiber with an electrowetting lens head to allow remote interrogation the vagus nerve with a bench top (i.e. portable) laser system. Integration of miniature (1mm diameter) scale electrowetting electrically tunable optics with an optical fiber-based imaging system will enable two-photon fluorescence imaging of neuron activity by readout of a fluorescent indicator. We will work with our collaborators in the field of pancreatic research to test, refine and demonstrate our ability to activate/report from in-vitro mouse vagus nerves and to see if we can control and/or sense pancreatic responses in the absence of other responses, such as a change in heart rate, using targeted neuro-modulation of specific axons in the vagus in in-vivo transgenic mice experiments.

Project Abstract We propose to develop a chronically implantable, all optical, optogenetic nerve interface that can non- invasively, optically neuromodulate individual axons of nerves in the parasympathetic or peripheral nervous system. The proposed interface would benefit treatment of human disease and disabilities related to the thoracic and abdominal organs and systems innervated by the cervical vagus nerve, such as epilepsy and metabolic disorders. We propose to optically interface from afferent/efferent axons in these nerves with the goal of modulating organs or brain circuits innervated by them. The bidirectional optical neural interface technology will utilize the capabilities of optogenetics enabled through viral vector transfection of afferent and/or efferent neurons with genetically targeted, optically activated reporter proteins and opsins. Our central premise is that we can use optics to communicate with axons in a nerve. For optical approaches to work we need to convert action potentials into an optical signal. This can be done using genetically encoded calcium indicators or other voltage sensitive proteins that change their fluorescent properties upon action potential generation in a neuron. Because nerves do not naturally express optical proteins, we will work with transgenic mice that express these proteins and use these mice models to refine our system before making it available for other researchers to use. We aim to develop a compact, bench-top optical system that can be shared with other research labs to provide the unique ability of being able to interrogate specific fascicles and axons within the nerve. In the future, this technology has potential for translation to human clinical applications. The technology in the proposal is ambitious, but we have formed an outstanding team of cell biologists, neuroscientists, biomedical, electrical, and mechanical engineers. The team has an excellent track record of successful collaborations on multiple grants and publications.