Jian Yang - US grants

The long-term objective of this research is to understand the molecular basis of the physiological functions of inwardly rectifying K+ (IRK) channels. IRK) channels, IRK channels are abundant in brain, heart, kidney, and pancreas. They are highly selective for K+ ions and conduct more K+ into cells than out of them, a property called inward rectification that underlies their crucial role in maintaining the resting membrane potential and regulating neuronal excitability, heart beat, and hormone secretion. Malfunction of IRK channels is known to cause neurological disorders in animal models. Thus, elucidation of the molecular mechanisms of ion permeation and inward rectification is important for understanding the physiological functions of inwardly rectifying K+ channels under normal conditions and in disorders such as epilepsy, arrhythmia, and diabetes. This research project focuses on a strongly rectifying channel, IRK1, that is abundant in both brain and heart. We propose to investigate the structural features of the ion permeation pathway, or pore, and the molecular determinants of K+ selectivity and inward rectification. Genetically altered IRK1 channels will be expressed in Xenopus frog oocytes and studied electrophysiologically using two-electrode voltage- clamp and patch-clamp. The specific aims are: (1) to map the structural domains involved in forming the internal portion of the channel pore, where cytoplasmic cations such as Mg2+ and polyamines bind to produce inward rectification; and to identify amino acid residues lining the ion conduction pathway in the H5 pore loop, which most likely forms the K+ selectivity filter and external entryway of the channel pore; (2) to study the molecular mechanism of K+ selectivity using the unnatural amino acid mutagenesis method: and (3) to localize amino acid residues in the inner pore that contribute to Mg2+ and polyamine binding; and to study the biophysical mechanism of interactions between permeant K+ ions and the blockers. Results from this research will help us understand better in general how inwardly rectifying K+ channels function. They will also allow us to gain better insight into the molecular architecture of the channel pore, which may help in the development of pharmacological agents directed at these channels.

[unreadable] DESCRIPTION (provided by applicant): Ion channels are crucial for diverse physiological functions, ranging from muscle contraction to hormone secretion, rhythmic beating of the heart to signaling in the brain. They selectively conduct ions across the cell membrane in response to changes in membrane potential or binding of a ligand. This research proposal seeks to understand the molecular and biophysical basis of ion permeation and gating of two types of ion channels, the inwardly rectifying K+ (Kir) channels and voltage-gated Ca2+ channels, both of which are abundant in heart and brain. Kir channels conduct less K+ during membrane depolarization due to block by cytoplasmic Mg2+ ions and polyamines and are regulated by a membrane phospholipid, PIP2. They play an important role in maintaining the resting membrane potential and regulating neuronal excitability, heart beat and hormone secretion. Voltage-gated Ca2+ channels mediate Ca2+ entry into cells in response to membrane depolarization and are vital for heartbeat and neurotransmitter release. We will use a combination of molecular biological, chemical and electrophysiological methods to investigate the structural features of the ion permeation pathway and mechanisms of channel gating. The proposed projects are: (1) to study the mechanism by which intracellular Mg2+ ions induce subconductance levels in Kir channel; (2) to examine the activation gate and conformational changes associated with PIP2 regulation of Kir channels; (3) to identify structural elements and amino acids that form the inner pore of Ca2+ channels; (4) to explore the molecular architecture of the selectivity filter including its size and the spatial organization of the four glutamates that are critical for Ca2+ selectivity and permeation; (5) to examine the activation gate and conformational changes associated with voltage-dependent activation of Ca2+ channels.Mutations in both Kir and Ca2+ channels have been found to cause neurological disorders in humans. Studies on the molecular mechanisms of ion permeation and gating of these channels are therefore important for understanding their physiological functions under normal conditions and in disorders such as arrhythmia, epilepsy and migraine. They may also help in the development of pharmacological agents directed at these channels.

DESCRIPTION (provided by applicant): Voltage-gated Ca2+ channels (VGCCs) mediate Ca2+ entry into neurons and are vital for brain functions. Mutations in VGCCs cause a variety of neurological disorders, including ataxia, migraine and epilepsy. The activity of VGCCs is constantly regulated by neurotransmitters and hormones in response to various internal and external stimuli, and such regulation profoundly affects Ca2+ signaling inside cells and hence brain functions. This research proposal seeks to understand the molecular and cellular mechanisms of regulation of N- and P/Q-type of VGCCs channels by phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane lipid critical for signal transduction, cytoskeletal organization and membrane trafficking. Our preliminary studies show that PIP2 exerts two distinct and opposing actions on the P/Q-type Ca2+ channels. On the one hand, PIP2 produces a strong voltage-dependent inhibition by altering the voltage-dependence of channel activation, an effect that can be prevented and reversed by PKA phosphorylation. On the other hand, PIP2 is crucial for maintaining the activity of the channels in intact cells. Furthermore, we discovered a mutation on a P/Q-type channel that dramatically reduces rundown of channel activity in excised membrane patches. We will build on these findings and carry out three areas of research. (1) We will investigate the molecular events and mechanisms underlying the dual actions of PIP2. Biochemical and site-directed mutagenesis studies will be used to identify regions and amino acids in the P/Q-type channel that are involved in PIP2 binding. (2) We will study the molecular mechanism of Ca2+ channel rundown. Specifically, we will investigate how the mutation mentioned above reduces rundown and whether its effect is linked to PIP2. (3) We will test the proposal that some well-known signaling pathways that regulate VGCCs do so by altering PIP2 interactions with the channels. Specifically, we will investigate whether G proteins inhibit Ca2+ channels by increasing the affinity of the channel for PIP2. This research may not only enhance our understanding of the complex regulatory pathways for VGCCs but also provide valuable information for the development of potential therapeutic reagents that target these pathways

High voltage-activated (HVA) calcium channels are essential for diverse biological processes ranging from gene regulation and cell growth to neurotransmitter release and muscle contraction. They are made up of several subunits, including alphal, alpha2-delta and beta. Although the beta subunit is only an auxiliary subunit, it is essential for trafficking the channel complex to the surface membrane and for the proper function of the channel. Other signaling proteins, such as G proteins and Rem/Rad/Gem(Kir) (RGK) family of small GTPases, regulate the activity of HVA calcium channels by either indirectly or directly interact with the beta subunit. Thus, the beta subunit is crucial for regulating the magnitude and kinetics of calcium signaling in excitable cells. Our group and two other groups have recently obtained high-resolution crystal structures of the beta subunit in complex with its primary binding partner in the alpha 1 subunit. This research proposal will combine x-ray crystallography, biochemistry and electrophysiology to further study the structure and function of the beta subunit and its interactions with other proteins, using the new structures as a blueprint. We will study: (1) the structural and biophysical mechanisms of regulation of HVA calcium channels by the beta subunit; (2) the interplay between regulation of HVA calcium channels by the beta subunit and G protein beta-gamma subunit; and (3) the structural and biophysical mechanisms of regulation of HVA calcium channels by the RGK GTPases. Mutations in the beta subunit cause human diseases such as idiopathic generalized epilepsy and episodic ataxia and a mutation in a RGK GTPase has been linked to congestive heart failure. These studies therefore may not only provide new insights into the basic mechanisms of calcium channel function and regulation but also new therapeutic strategies for treating neurological and cardiovascular diseases.

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand the molecular and structural mechanisms of the function of transient receptor potential (TRP) channels. In particular, we will focus on the canonical TRP (TRPC) channels, which are present in blood vessels, heart, lung, kidney and brain and are involved in regulating diverse biological processes ranging from neural and cardiovascular functions to sexual behavior. The seven members of the TRPC channel family conduct calcium into cells and are in turn regulated by intracellular calcium. All TRPC proteins interact directly with calmodulin (CaM), a ubiquitous calcium sensing protein, which may mediate calcium-dependent feedback regulation of TRPC channel activity and function. Our specific aims are to solve the high-resolution crystal structure of the complex of CaM and each of the four putative CaM-binding sites in TRPC proteins and to study the molecular mechanism and functional impact of CaM binding to each site. Our central hypothesis is that CaM forms distinct interactions with each TRPC site and these interactions regulate channel properties and functions in a channel-specific fashion. Mutations in TRPC channels have been linked to focal and segmental glomerulosclerosis, a hereditary kidney disorder leading to renal failure, and the abundance of TRPC channels in airway smooth muscles and blood vesicles suggest that their malfunction could play a role in asthma, chronic obstructive pulmonary disease, hypertension and various types of cardiovascular diseases. Thus, our studies will not only provide a better understanding of the function and regulation of these biologically important ion channels but also facilitate the development of new therapeutic strategies for the treatment of a host of human diseases. PUBLIC HEALTH RELEVANCE: TRPC channels are found in blood vessels, heart, lung, kidney and brain and are involved in regulating numerous biological processes ranging from neural and cardiovascular functions to sexual behavior. Malfunction of these channels may cause human diseases such as asthma and various types of cardiovascular diseases. A better understanding of how these channels work may provide new ways to treat a host of human diseases.

DESCRIPTION (provided by applicant): Approximately 0.1% of the world population is affected by autosomal dominant polycystic kidney disease (ADPKD), which is one of the most common genetic diseases in humans. This disease is caused by mutations in two polycystin proteins, PKD1 and TRPP2. PKD1 is a plasma membrane receptor probably involved in cell-cell and cell-matrix interactions. TRPP2 is a Ca2+permeable nonselective cation channel present in the endoplasmic reticulum as well as on the plasma membrane. In kidney cells, they form a cell surface complex that may function as a mechanosensitive Ca2+conducting ion channel that can be regulated by fluid flow in the kidney tubule. The association of TRPP2 and PKD1 is mediated, at least in part, by a coiled coil domain in the cytoplasmic C terminus of both proteins. TRPP3, related to but distinct from TRPP2, also forms a Ca2+permeable nonselective cation channel, and deletion of its gene in mice is linked to fatal kidney defects. Its cytoplasmic C terminus also contains a coiled coil domain. Recently, it has been found that TRPP3 associates with a PKD1-like protein named PKD1L3 to form the receptors for sour taste in the tongue. Our long-term objective is to understand the structural, molecular and cellular mechanisms of the function and regulation of polycystins. The immediate goal is to investigate the assembly and stoichiometry of the TRPP2/PKD1 and TRPP3/PKD1L3 complexes. We propose to solve the crystal structure of the putative coiled coil domain of both complexes, determine the subunit stoichiometry of both complexes in cells, investigate the importance of the coiled coil domain interaction in the assembly of both complexes and in the PKD1 regulation of TRPP2 activity and PKD1L3 regulation of TRPP3 activity. We also propose to test the hypothesis that PKD1 and PKD1L3 directly participate to form the channel pore in the TRPP2/PKD1 complex and the TRPP3/PKD1L3 complex, respectively. This research will enhance our understanding of the molecular basis of polycystin function. PUBLIC HEALTH RELEVANCE: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic diseases in humans and is caused by mutations of polycystin complexes in kidney cells. The goal of this research is to understand how these complexes are formed and how they function.

DESCRIPTION (provided by applicant): Voltage-gated calcium channels (VGCCs) control and regulate numerous physiological processes, ranging from muscle contraction, heartbeat, neural communication and hormone secretion to cell differentiation, motility, growth and death. As such, their activity is tightly regulated by diverse molecules and pathways, including protein kinases and phosphatases, G proteins, calcium and calmodulin, SNAREs, and phospholipids. We have uncovered a new paradigm for VGCC regulation: The pore-forming a1 subunit of neuronal surface L-type VGCCs is proteolytically cleaved in the cytoplasmic loops (mainly the II-III loop but also the I-II loop) connecting the four homologous repeats, this cleavage is activity-dependent, and the cleaved fragments (called hemi-channels) physically separate. This novel form of channel regulation may significantly impact L-type calcium channel activity, calcium signaling, and gene transcription in neurons. We propose to study the signaling molecules and pathways, the dynamics, and the functional consequences of mid-channel proteolysis, and the fate and function of the resulting hemi-channels, using a combination of biochemistry, proteomics, fixed cell and live cell imaging, and electrophysiology. As intracellular calcium signaling is severely altered in aging neurons and age-related neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, we will investigate whether mid-channel proteolysis and hemi-channel separation are perturbed in these physiological and pathological processes. We will also expand our studies to N- and P/Q-type calcium channels, which mediate neurotransmitter release. These studies will provide mechanistic insights into a hitherto ill-studied form of VGCC regulation and may lead to the development of new therapeutic strategies for calcium homeostasis-related diseases.

Restoring living tissue functionality via tissue engineering is crucial for transformative advances in medicine. Tissue engineering materials must be biocompatible and often biodegradable in a controlled manner. For example, severed peripheral nerves can regrow, but new projections must be properly nourished and guided via tissue scaffolds. Scaffolds must have the right morphology for cell growth, the right transport properties for nourishing cells, and the right mechanical properties to stay compliant and integral during degradation and tissue regeneration. Biodegradable scaffolds are appealing because they need not be surgically removed; but they are effective only if degradation is synchronized with nerve regrowth. This is but one of many examples illustrating the extraordinary challenges in tissue engineering. This award will yield a multi-scale approach based on physics, mathematics, polymer chemistry, and image analysis to predict and interrogate evolving transport and mechanical properties of porous polymeric scaffolds during programmed enzymatic degradation. The contribution of the project to the advancement of mechanics is a new methodology to model, and thus understand, the behavior of multi-functional materials with evolving microstructure like those in nerve tissue engineering. An educational component is included to attract underrepresented minorities to engineering via level-appropriate workshops on applications of mechanics in neuroscience, and by involving undergraduates in the creation of coursework for courses in brain biomechanics.

Biodegradable tissue engineering systems are deformable chemically-reacting porous mixtures with complex fluid-structure interaction. The project integrates specific existing averaging techniques with an original fluid-structure interaction approach to determine the coupled mechanical and transport properties of degrading porous polymer networks subjected to large deformation and mechanical loadings. The model system of relevance to the project is crosslinked urethane-doped polyester, a promising scaffold material for nerve regeneration with highly controllable porosity. This material will be modeled as a random polymer network. Samples will be analyzed via electron microscopy to quantify the network's morphology. Microscopic-level transport and mechanical properties will be determined via a statistical characterization of the polymer network structure. This process will define microstructurally accurate representative volume elements whose evolution can then be analyzed via a novel finite element fluid-structure interaction-based homogenization procedure for evolving microstructure due to degradation. This numerical scheme will yield effective mechanical and transport properties at the mesoscale as a function of degradation. A crucial advancement in mechanics is the framing of the homogenization problem as a fluid-structure interaction problem, by extending the immersed finite element method (a state-of-the-art fluid-structure interaction computational approach) to account for fluid flow through bodies with evolving microstructure. The project includes experiments to validate predicted properties. Material samples and full-scale scaffold at different stages of degradation will be characterized in terms of morphology, elastic moduli, and diffusivity, and these properties compared to corresponding numerical estimates.

Chromatic two-photon fluorescence microscopy using band-shifting imaging probes Two-photon fluorescence microscopy has emerged as one of the most important imaging modalities for biological research and medical diagnosis. Although many techniques exist for realizing high speed two-photon imaging in the lateral directions, the axial imaging speed is still often limited by the slow mechanical scanning of the objective lens or the specimen, presenting a significant challenge for monitoring fast biological processes at multiple depths as well as in 3D and the development of miniature endoscopy. To overcome this limitation, here a new spectrally encoded two-photon imaging technique is proposed using band-shifting imaging probes, which can enable parallel axial imaging. Specifically, different excitation wavelengths are focused onto different axial positions (through purposely introduced chromatic aberration) to excite two-photon fluorescence from the band-shifting imaging probes, which shift the emission band when the excitation wavelength varies. As such, the fluorescence signals at different axial positions are spectrally encoded to exhibit different spectral bands, and can thus be imaged in parallel by using a spectrometer or arrayed wavelength-resolving detectors. The proposed band-shifting imaging probes will be synthesized, optimized, and used for cellular labeling. Systematic characterization on the molecular structures, molecular weight, photophysical and two-photon properties will be performed. The proposed chromatic two-photon imaging system will be designed, developed, and optimized. System metrics including the mapping relationship between the axial position and fluorescence band shift, the axial imaging range, and spatial resolutions will be characterized. The proposed method will be demonstrated and validated by performing imaging of cells and tissue phantoms as well as by in vivo imaging studies of a colorectal cancer xenograft model.