The Yan Lab is interested in understanding structure, function and regulation of mammalian ion channels related to pain, neurological diseases and cancer. Ion channels are membrane protein complexes that translocate ions across cell or organelle membranes, underlying a broad range of the most basic physiological processes from nerve and muscle excitability, to membrane potential setting, pH/cell volume regulation, secretion and absorption. Ion channels have long been key therapeutic targets in disease intervention and pharmaceutical drug development because of their direct involvement in diverse diseases, vulnerability to small molecular modulation, and accessibility for direct activity measurement on cell membranes by patch-clamp recording from whole cell to single molecule levels. We are interested in multiple research directions.
1. Functional proteomics of native mammalian ion channel signaling complexes
Functional proteomics is aimed to understand proteins’ biological function and their regulation by studying protein-protein interactions within and among multiprotein complexes through affinity purification and mass spectrometric analysis. Latest proteomic research shows that native ion channels commonly exist as a large functional unit of “signaling complex” consisting of the ion-conducting pore subunits, variable peripheral auxiliary subunits, and interacting protein partners. Because of the low abundance of most native ion channel proteins in mammalian cells, the purified native ion channel complexes are commonly contaminated by an overwhelming number and/or amount of non-specifically co-purified proteins, which makes the downstream mass-spectrometric and functional analyses very difficult. We overcome these hurdles by increasing affinity-purification efficiency and employing quantitative proteomic analysis to reduce and effectively screen out contaminants. We have identified: (i) a new family of BK channel regulatory proteins, designated as BK channel g subunits, which are a group of leucine-rich repeat (LRR) containing membrane proteins, LRRC26 (γ1), LRRC52 (γ2), LRRC55 (γ3), and LRRC38 (γ4); (ii) NMDA receptors as BK channel interacting partners forming the glutamate-activated K+ channel complexes in brain; (iii) Lsm12 as a novel NAADP receptor and a two-pore channel (TPC) auxiliary protein necessary for NAADP-evoked Ca2+ release.
2. Molecular mechanisms and function of BK channels
The large-conductance, Ca2+ and voltage-activated K+ channel (BK, also termed as BKCa, Maxi-K, KCa1.1 or Slo1) is a unique member of the mammalian K+ channel family, which has the largest single channel conductance and is dually activated by membrane voltage and intracellular Ca2+. BK channels consist of pore-forming, voltage-, and Ca2+-sensing α and auxiliary β and γ subunits. The auxiliary γ subunits display an exceptionally large range of capabilities in shifting the BK channel’s voltage dependence of activation towards the hyperpolarizing direction by 145 – 20 mV in the absence of Ca2+. BK channels are innovative drug targets for disorders of almost every organ system. BK channels play a variety of physiologically important roles, such as neuronal firing and neurotransmitter release, frequency tuning of auditory hair cells, hormone secretion, and contractile tone of smooth muscles. Defects in BK channels can cause epilepsy and paroxysmal dyskinesia, high blood pressure, urinary incontinence, and erectile dysfunction. BK channels possess many biophysical features that make them an ideal system for studying allosteric mechanisms of channel gating and modulation by regulatory proteins and drugs. We are interested in studying: 1) the central pore gating mechanisms of BK channels; 2) the molecular mechanisms of BK channel modulation by auxiliary γ subunits; 3) the physiological and pathological roles of BK channel auxiliary γ subunits in learning and memory, movement and coordination, and neurological disorders; 4) the molecular mechanisms and physiological roles of BK channel-NMDA receptor coupling complexes in brain.
3. Molecular mechanisms and function of NAADP-gated Ca2+ release channel complexes
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 signaling is implicated in a broad range of cellular and physiologic processes such as neurotransmitter release and neurosecretion, membrane excitability, autophagy, exocytosis, fertilization, contraction of cardiac and smooth muscles, cell differentiation, insulin secretion, and glucose uptake and in many diseases, including lysosomal storage diseases, diabetes, autism, and cardiovascular, blood, and muscle diseases. Despite the importance of NAADP-evoked Ca2+ signaling, the molecular basis and function of NAADP-gated Ca2+ release channel complexes remains largely unclear. Tow-pore channels (TPCs) have been considered as the key endolysosomal cation channels responsive to NAADP stimulation for intracellular Ca2+ release. With immobilized NAADP–based affinity purification and quantitative proteomic analyses of NAADP and TPC interacting proteins, we identified Lsm12 to be a shared interacting partner of NAADP, TPC1, and TPC2. Lsm12 directly binds to NAADP via its Lsm domain, colocalizes with TPC2, and mediates the apparent association of NAADP to isolated TPC2 or TPC2-containing membranes. Lsm12 is essential and immediately participates in NAADP-evoked TPC activation and Ca2+ mobilization. Our findings thus reveal a putative RNA-binding protein functioning as an NAADP receptor and a TPC regulatory protein and provide a new molecular basis for understanding the mechanisms and function of NAADP signaling. We are further interested in studying: 1) the molecular mechanisms of Lsm12’s function as an NAADP receptor; 2) the molecular mechanisms of TPC channel activation by NAADP-Lsm12; 3) the physiological and pathological roles of Lsm12-mediated NAADP signaling; 4) identification of other novel proteins including ion channels involved in NAADP signaling.