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 (channelopathy), 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 two research directions.
1. Regulatory mechanism and physiological roles of BK channel γ-subunits.
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+ ([Ca2+]i), playing an integrative role in regulation of cellular excitability and calcium signaling. BK channels are critically involved in diverse physiological processes 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.
With proteomic and electrophysiological approaches, we recently identified a new family of BK channel regulatory proteins, designated as BK channel γ-subunits (Yan J and Aldrich RW, 2010 Nature 466:513-6; Yan J and Aldrich RW 2012 PNAS 109: 7917-22). They are a group of leucine-rich repeat (LRR) containing membrane proteins, LRRC26 (γ1), LRRC52 (γ2), LRRC55 (γ3), and LRRC38 (γ4). The γ1-subunit causes an unprecedented large negative shift (~ -140 mV) in voltage dependence of the BK channel activation, conferring BK channels with an unusual capability to activate at resting voltages and [Ca2+]i levels in excitable and non-excitable cells. The other three γ-subunits also produce marked shifts in the BK channel’s voltage dependence of activation in the hyperpolarizing direction by approximately 100 mV (γ2), 50 mV (γ3), and 20 mV (γ3), respectively, in the absence of calcium. They show distinct expression in different human tissues (γ1 and γ4 mainly in secretory glands, γ2 in testis, and γ3 in brain) and potentially regulate the channel’s gating properties over a spectrum of different tissues or cell types.
Structural models of BK channel α- and γ-subunits and the modulatory effects of γ-subunits in the absence of calcium. α-subunit was modeled with structures of Kv channel (PDB 2R9R) and BK intracellular domain (PDB 3MT5 & 3NAF). γ-subunit was modeled with hagfish VLR-B (PDB 2O6S) and mouse TLR4 (PDB 2Z64).
To elucidate regulatory mechanism and physiological roles of the BK channel γ-subunits, we are currently interested in studying (i) structural mechanism of BK channel activation by the γ-subunits, (ii) the transcriptional regulation of the γ1-subunit’s differential expression, and (iii) neurological role of the γ3-subunits in brains. The findings from these research projects will help in understanding the gating mechanism of BK channel activation and provide structural and molecular basis for rational drug design and manipulation of BK channel activity with great therapeutic potential.
2. Functional proteomics of native mammalian ion channel signaling complexes
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 have been identified, which are encoded by more than 300 human genes and account for about ~1.5% of human proteome. Since 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 new ion channel principle or regulatory proteins.
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. Because of the low abundance of most native ion channel proteins in mammalian cells, a major limitation of the functional proteomic approach is that the purified native ion channel complexes are commonly contaminated by a large number and overwhelming amount of non-specifically co-purified proteins, which makes the downstream mass-spectrometric and functional analyses very difficult. Efficient affinity-purification, carefully chosen negative controls and high resolution nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) will be employed to dissect the ion channels signaling complexes and explore new ion channel proteins.