Jiusheng Yan, Ph.D. - US grants

DESCRIPTION (provided by applicant): Large conductance, calcium and voltage-activated potassium (BK) channel is a unique member of potassium channel family, which has the largest single channel conductance and is dually activated by voltage and cytosolic free Ca2+ ([Ca2+]i), consisting of the pore-forming, voltage and Ca2+-sensing -subunits (BK) either alone or in association with tissue specific regulatory -subunits (1 - 4). BK channels are generally hard to open, requiring coincident membrane depolarization and [Ca2+]i rise for activation in vivo. However, with proteomic and electrophysiological approaches, we have recently identified a novel BK channel auxiliary subunit, leucine-rich repeat (LRR) containing membrane protein LRRC26 that causes an unprecedented large negative shift (~ -150 mV) in voltage dependence, allowing BK channel activation at even near resting voltages and calcium levels in excitable and non-excitable cells. LRRC26 represents a new family of BK channel auxiliary subunits, which is structurally distinct from the four known -subunits and designated as a -subunit. Like the presence of multiple tissue specific BK channel -subunits with different modulatory functions, we hypothesized that there exist other members of the LRRC26-like -subunits, which differentially modulate BK channels over a wide range of different cell types. The following two specific aims are designed to delineate the family members and to define the structural and functional features of the BK channel -subunits: 1) Identify and characterize new members of BK channel -subunits; 2) identify the structural determinants for modulatory function of the BK channel -subunits. We have identified five LRRC26 paralogs, LRRC38, LRRC52, LRRC55, LRTM1 and LRTM2, from protein database. Experiments are designed to determine the modulatory effects of these LRRC26 paralogs on BK channel function, and to identify key structural elements responsible for a LRR protein to be a BK channel modulator. Overall, the proposed research is designed to identify new family members and to define the structural and functional features of the BK channel -subunits. The findings from the proposed research may establish a broad LRR family of ion channel auxiliary subunits, gain an in-depth understanding of the LRRC26's unique capacity in BK channel modulation, and provide new protein targets for BK channel related disease treatment and drug development.

DESCRIPTION (provided by applicant): The large conductance, calcium- and voltage-activated potassium (BK) channel is a unique member of the potassium channel family, which has the largest single channel conductance and is dually activated by voltage and cytosolic free Ca2+. BK channels consist of the pore-forming, voltage- and Ca2+-sensing -subunits (BK) either alone or in association with the tissue-specific regulatory subunits including the four previously known ?-subunits. We recently identified a novel BK channel auxiliary subunit, a leucine-rich repeat (LRR) containing membrane protein LRRC26, which causes an unprecedented large negative shift (~ -150 mV) in voltage dependence of channel activation by greatly enhancing the allosteric coupling between the voltage-sensor activation and the channel's closed-open transition, allowing BK channel activation at even near resting voltages and calcium levels in excitable and non-excitable cells. We have additionally identified three LRRC26- like paralogous proteins that modify the BK channel's voltage dependence of activation to different extents. LRRC26 and its paralogous LRR proteins are structurally and functionally distinct from the ?-subunits and are thus collectively designated as a family of BK channel ?-subunits. Three specific aims are designed to determine the physiological relevance and molecular mechanisms of BK channel regulation by these auxiliary ?-subunits: 1) determine the physiological and functional expression of the BK channel ?-subunits in human tissues and cells; 2) determine the biochemical mechanisms of BK channel modulation by the ?-subunits; 3) determine the posttranslational regulation of the ?-subunits' modulatory functions. Molecular biological, biochemical and electrophysiological experiments will be performed to achieve the proposed aims. Overall, the proposed research in this grant application is designed to systematically investigate these auxiliary LRR proteins for their physiological relevance and the underlying molecular mechanisms of channel modulation. The findings from the proposed studies will establish the physiological relevance of a new family of BK channel auxiliary subunits, and provide an in-depth understanding of the molecular mechanisms governing the LRRC26 and its paralogs' unique capacity in shifting the voltage dependence of a voltage-gated ion channel. These studies will thus offer a new molecular basis for an understanding and exploration of the ubiquitously expressed BK channel's diverse physiological functions, and help in creation of novel reagents and therapeutics to rationally manipulate BK channel activity. PUBLIC HEALTH RELEVANCE: The findings from the proposed research will provide a new molecular basis for an understanding and exploration of the ubiquitously expressed BK channel's diverse physiological functions, and help in creation of novel reagents and therapeutics to rationally manipulate BK channel activity.

Intracellular Ca2+ signaling via changes in cytosolic Ca2+ concentration controls a wide range of cellular and physiologic processes. Ca2+ mobilization from intracellular stores mediated by second messengers is an important source of cytosolic Ca2+. Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-mobilizing second messenger identified to date; it uniquely mobilizes Ca2+ from acidic endolysosomal organelles. NAADP has been shown to be effective in evoking Ca2+ release in a multitude of different mammalian cells and defects in NAADP signaling are now being implicated in many diseases. Despite the importance of NAADP-evoked Ca2+ signaling, the molecular identities of the NAADP receptors and Ca2+- release channels involved in this process have yet to be unequivocally defined. Accumulated evidence indicates that endolysosomal two-pore channels (TPCs) might participate in NAADP-evoked Ca2+ release. However, recent direct patch-clamp recordings of TPC channel currents in endolysosomes showed that TPCs were Na+-selective channels with very limited Ca2+ permeability, and the channel activity was not sensitive to NAADP. Currently, a unifying hypothesis about the role of TPCs in NAADP signaling is that TPCs are not directly involved in NAADP binding but are part of the NAADP receptor/Ca2+-release channel complex, in which the molecular identities of other key proteins remain unknown. These new findings and speculations warrant an exploration of additional proteins that may be important for NAADP-evoked Ca2+ signaling. In the proposed study, we will use both TPCs and NAADP as baits to isolate their interacting partners from mammalian HEK293 and SKBR3 cells and then use unbiased quantitative proteomic analysis and functional assays to screen and identify novel proteins important for NAADP-evoked Ca2+ signaling. We will pursue the following two specific aims. Specific Aim 1. Identify novel TPC-interacting proteins important for NAADP-evoked Ca2+ signaling. We aim to systematically determine the interactome of TPCs using a SILAC-based quantitative proteomic approach with advanced mass spectrometry. We will then perform Ca2+ imaging functional assays on identified TPC-interacting proteins to identify novel proteins important for NAADP-evoked Ca2+ signaling. Specific Aim 2. Identify NAADP-interacting proteins important for NAADP-evoked Ca2+ signaling. To directly target the NAADP receptor, we will perform affinity purification of NAADP-interacting proteins from HEK293 and SKBR3 cells using immobilized NAADP and its analogue and then employ quantitative proteomic analysis and functional assays to identify novel proteins important for NAADP-induced Ca2+ release. The proposed studies are designed to use systematic approaches to unbiasedly screen and identify novel proteins important for NAADP-evoked Ca2+ release. Our results will likely generate a breakthrough in the molecular basis and mechanisms of NAADP signaling by identifying novel proteins that could serve as NAADP receptors, Ca2+- release channels, or regulatory proteins necessary for NAADP-evoked Ca2+ release. 1

Project Summary: Intracellular Ca2+ signaling via changes in cytosolic Ca2+ concentration controls a wide range of cellular and physiologic processes. Ca2+ mobilization from intracellular stores mediated by second messengers plays a critical role in regulation of cytosolic Ca2+ levels. Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-mobilizing second messenger identified to date; it uniquely mobilizes Ca2+ from acidic endolysosomal organelles. NAADP has been shown to be effective in evoking Ca2+ release in a multitude of different mammalian cells and defects in NAADP signaling are now being implicated in many diseases. Despite the importance of NAADP-evoked Ca2+ signaling, the molecular identity of the NAADP receptor remains elusive and the Ca2+-release channels involved in this process have yet to be unequivocally defined. Accumulated evidence indicates that endolysosomal two-pore channels (TPCs) play an important role in NAADP-evoked Ca2+ release. However, strong evidence also suggests that TPCs are not NAADP receptors. Currently, a unifying hypothesis is that TPCs are the key channels responsible for NAADP-evoked Ca2+ release but that their NAADP sensitivity arises from some unknown NAADP-binding accessory protein. To identify the elusive NAADP receptor, we used both TPCs and NAADP as bait to isolate their interacting partners from mammalian HEK293 and SKBR3 cells and then used an unbiased quantitative proteomic analysis and a Ca2+- imaging functional assay to screen and identify novel proteins that are important for NAADP-evoked Ca2+ release. We have identified an Lsm12 protein to be an interacting protein of both NAADP and TPCs. With Lsm12-knockout cells, we found that Lsm12 mediates the interaction between NAADP and TPCs and is essentially required for NAADP-evoked Ca2+ release in HEK293 cells. We hypothesize that Lsm12 is essential and directly involved in NAADP-evoked Ca2+ release by functioning as a NAADP receptor and/or a TPC regulatory or auxiliary protein. We will pursue the following 3 specific aims: 1) test the hypothesis that Lsm12 is essential and directly involved in NAADP-evoked ca2+ release; 2) test the hypothesis that Lsm12 is a NAADP receptor; and 3) test the hypothesis that Lsm12 is a regulatory or auxiliary protein of TPCs. We will achieve these goals by using multidisciplinary approaches from molecular biology, cell biology, protein biochemistry, and electrophysiology. Findings from the proposed research will provide a breakthrough that will advance our understanding of the molecular basis and mechanisms of NAADP-evoked Ca2+ release, and facilitate the development of new drugs for this important Ca2+ signaling process.

Large-conductance, calcium- and voltage-activated potassium (BK) channels play a variety of physiologically important roles, are innovative drug targets for disorders of almost every organ system, and possess biophysical features that make them an ideal system for studying allosteric mechanisms of channel function (gating) by voltage and ligands and modulation by drugs. The BK channel is a unique member of the potassium channel family, characterized by exceptionally large single-channel conductance and dual activation by two physiological signals, membrane voltage and intracellular free calcium. A variety of experimental evidence indicates that BK channels lack the intracellular bundle-crossing gate that is present in many other potassium channels. Thus the opening and closing of the BK channel pore during activation must be controlled by other mechanisms. Recent determination of the 3D structure of the complete BK channel from Aplysia californica at a near-atomic resolution provides a new structural basis for understanding these mechanisms. The structures not only confirm that the lack of a bundle-crossing gate, but suggest novel mechanisms of BK channel activation mediated by state-dependent interaction among amino acids in the deep pore and selectivity filter regions. We hypothesize that the BK channel activation gate is located within the selectivity filter and/or deep-pore. We have made progress towards testing this hypothesis by establishing methods to determine the relationship between activation and selectivity filter inactivation and analyzing the structure-function relationship of BK channel pore residues. With the newly available structural information and novel tools that we have developed, including concatenated tandem subunit constructs to restrict mutations to individual BK channel subunits within the tetrameric channel, we are now poised to determine the pore gate localization and central channel pore gating mechanisms. We propose to pursue the following three specific aims to elucidate the pore-gating mechanisms of BK channels: 1) determine the properties and mechanisms of selectivity filter gating in BK channels; 2) determine the role of the deep-pore residues and their interactions in BK channel gating; and 3) define the location of the activation and inactivation gates by determining the state- dependent accessibility of the selectivity filter and deep pore to Cys-modifying reagents. Overall, the proposed research is designed to investigate systematically the central pore-gating mechanisms of BK channels. The findings from the proposed studies will deepen our understanding of molecular mechanisms of BK channel activation by voltage and calcium and facilitate development of novel therapeutic reagents targeting BK channels for the treatment or prevention of neurobiological, cardiovascular, and other types of disorders and diseases.