Our research focuses on the structural and functional studies of ion channels and transporters, which regulate the flow of ions across the cell membrane. These proteins regulate many biological processes, such as the excitation of nerve and muscle cells, the secretion of hormones, and sensory transduction. Our approach combines membrane protein x-ray crystallography, to determine the three-dimensional structure of the ion-transporting proteins, and channel electrophysiology, to study the physiological functions of these proteins.
Ion channels are membrane proteins that form a pore through the hydrophobic lipid bilayers and allow for the free diffusion of ions down their electrochemical gradients. Two fundamental properties are central to ion channel function: ion selectivity, whereby only the passage of specific ions is allowed through the channel pore, and channel gating, where the opening and closing of the channel pore is regulated in response to a specific stimulus. Our research is aimed at understanding the molecular mechanisms of both channel selectivity and gating in tetrameric cation channels, the single largest family of ion channels. In these channels, four membrane-spanning subunits or domains form a central pore through which specific ions cross the cell membrane.
To study channel gating, we focus on a group of ligand-gated K+ channels that are regulated by a conserved ligand-binding domain, the RCK domain. This group includes the majority of prokaryotic K+ channels and the eukaryotic high-conductance Ca2+-gated K+ channels (BK or maxiK). We are using three RCK-regulated K+ channels as model systems for studying ligand specificity and ligand-induced conformational changes in K+ channels: MthK, a Ca2+-gated K+ channel from Methanobacterium thermoautotrophicum (Figure 1); GsuK, a nucleotide-gated Ca2+-inhibited K+ channel from Geobacter sulfurreducens; and the BK channel (hSlo1), a human high-conductance Ca2+-gated K+ channel.
Another goal of our research is to understand the structural basis of ion selectivity among tetrameric cation channels, using the NaK channel, a nonselective prokaryotic cation channel from Bacillus cereus, as a model system. Taking advantage of the extremely high-resolution crystal structures of NaK and its mutants that represent the ion conduction pores of both selective and nonselective cation channels (Figure 2), we aim to elucidate the basic principles of ion selectivity in two families of physiologically essential cation channels. One family is the nonselective, Ca2+-permeable cyclic nucleotide-gated (CNG) channels, whose functions are central to signal transduction in the visual and olfactory sensory systems, and the other is the K+ selective channel family.
While there is a long history of physiological work and a large body of functional and structural data on tetrameric cation channels that are localized to the plasma membrane, relatively little is known about organellar cation channels, partly because of the difficulty in directly measuring their activities in organellar membranes. Recently, we expanded our research of channel gating and selectivity to eukaryotic organellar two-pore channels (TPCs). Currently, there is an emerging research interest in the recently defined TPC channels due to their importance in endo/lysosome physiology. In human and animals, TPC channels regulate the ionic homeostasis and pH within lysosomes, set the lysosomal membrane potential and excitability, and may also regulate lysosomal Ca2+ release. Therefore, TPC channel functions directly or indirectly affect lysosome-mediated processes such as cellular degradation as well as catabolite export and trafficking, and defects of these processes can result in lysosomal storage diseases. Thus, understanding the molecular basis of TPC channel functions will provide basic, fundamental knowledge about many TPC-related lysosomal activities and diseases.
Distinct from ion channels, ion transporters can move ions across the plasma membrane against the electrochemical gradient. The study on ion transporters in the lab has been focused on the Na+/Ca2+ exchanger.
Na+/Ca2+ exchangers (NCXs) are membrane transporters that are central to the maintenance of the homeostasis of cytosolic Ca2+, which acts as signaling molecule for numerous, vitally important cellular processes; these include muscle contraction, cell mobility, fertilization, exocytosis, and apoptosis. NCX proteins use the downhill gradient of Na+ to extrude intracellular Ca2+ across the cell membrane against its chemical gradient. Several functional features of an NCX protein define its physiological roles: it can exchange Ca2+ and Na+ with a high-turnover rate; the ion-exchange process is electrogenic, with a stoichiometry of 3 Na+ for 1 Ca2+; and the exchange reaction is bidirectional, depending on the membrane potential and the chemical gradient of Na+ and Ca2+. Despite a large body of functional data, the structural mechanism underlying these functional features remains elusive. With the determination of a high-resolution structure of an NCX protein from Methanococcus jannaschii (NCX_Mj) (Figure 3), we now have a working model to elucidate the structural basis and mechanistic details of ion exchange in NCX.
This research is also supported by grants from the National Institutes of Health and the Welch Foundation.
As of March 3, 2016