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 (blockers or activators), and accessibility for direct activity measurement on cell membranes by patch-clamp recording from whole cell to single molecule levels. We are currently 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 functional 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. So far, more than 230 different human ion channels encoded by ~ 300 human genes have been identified; but only a very limited number of ion channel auxiliary proteins have been reported. Given that a large portion (~40%) of human proteins still don’t have a well-defined biological function, it is anticipated that a reasonable number of these function-unknown proteins may function as ion channel regulatory or even principle proteins. 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 are currently working on identification of novel ion channel proteins by functional proteomic analysis of multiple ion channels signaling complexes purified from brain tissues or cultured mammalian cells. As an example for this line of exploratory research direction, we have identified 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 (g1), LRRC52 (g2), LRRC55 (g3), and LRRC38 (g4). These auxiliary g 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+]i.
2. Biophysical and physiological studies on mechanisms of BK channel activation and regulation
The large-conductance, calcium and voltage-activated potassium 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 are ubiquitously expressed in different tissues and cells and their activities are regulated by a variety of endogenous modulators in vivo. 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 drugs. To elucidate the mechanisms of BK channel activation and regulation and facilitate creation of novel therapeutic reagents targeting BK channels, we are currently interested in studying: 1) the regulatory mechanisms and physiological roles of BK channel auxiliary g subunits; 2) the channel gating mechanisms; 3) how the BK channels are structurally and functionally coupled to Ca2+-permeable channels for effective channel activation by local Ca2+ flux in neurons.