Ion channels are ubiquitous proteins, vital to scores of cellular processes including cell membrane excitability and propagation of nervous stimuli. Structural defects within ion channel proteins alter channel function and in turn cause human disease.
The main focus of the lab is is to elucidate the mechanisms of gating, selectivity, and modulation of potassium channels.
The prokaryotic K+ channel KcsA is gated by intracellular protons. Using structure guided mutagenesis, we elucidated the molecular mechanism of proton sensing in this channel. We found that the sensor consists of a network of inter- and intra-subunit salt bridges and hydrogen bonds that stabilize the channels in a closed state at high pH. This network is disrupted at low pH as the amino acids change their ionization states, allowing the channel to open. We are further interested in the energetic contributions of the individual residues in the proton sensing region and looking into the effect these mutations of the proton sensor have on the gating kinetics. We approach these questions with lipid bilayer electrophysiology and X-ray crystallography.
MthK is a ligand-gated ion channel from bacteria for which we have (low resolution) structural information from x-ray crystallography. Ca2+ induced single channel or ensemble currents can be measured from purified protein and we can study questions such as – How is the channel conduction gate regulated by calcium? Where is the physical location of this conduction gate? How do pH and membrane voltage modulate the gating process? These questions are addressed by using molecular biology, biochemistry, x-ray crystallography, and electrophysiology.
MthK single channel recording In conditions that favor a highly open K+ channel, blocking events from a quaternary ammonium compound can be measured. The blocker binds to the pore and interrupts the flow of K+, yielding the long zero-current intervals (on the dashed line).
MthK M107I crystals
KcsA, like its eukaryotic counterparts, is highly selective for K+ over Na+. This was thought to arise due to a lack of a binding site in the selectivity filter. Using results from electrophysiology, X-ray crystallography and molecular dynamics simulations of the model K+ channel KcsA, we propose that Na+ (Li+) have a separate, novel binding-site within the K+ selectivity filter. We proposed that selective permeation from the intracellular side primarily results from a large energy barrier blocking filter entry for Na+ in the presence of K+. We are now exploring the question whether this mechanism also contribute to selective permeation from the extracellular side and whether there are additional small cation binding sites in the selectivity filter.
Cyclic nucleotide-modulated channels
Cyclic nucleotide-modulated channels are involved in the visual and olfactory signal transduction cascades. The function of these channels is modulated by cGMP or cAMP levels in the cell. These large ion channels have separate domains for specific functions, for example, the cyclic nucleotide binding domain (CNBD) binds the ligands that modulate open probability of the channels and the pore region enables ion permeation. We are interested in elucidating how these different domains interact to control channel function. For this we use a prokaryotic channel from Mesorhizobium loti called MloK1. A low resolution EM study of full-length MloK1 appears to support a fourfold symmetry of the CNBDs similar to their arrangement in eukaryotic HCN2 channel. At this point, the molecular mechanism of ligand modulation in MloK1 is still unknown.
Novel lipoprotein technologies for channel biophysics
Nanoscale apolipoprotein bound bilayers (NABBs) are soluble lipid nanoparticles that may be used as a membrane-mimetic system for membrane proteins that are not stable in detergents. Studies of purified ion channels have traditionally involved biophysical assays performed on either detergent-solubilized proteins – a non-native preparation, or using proteoliposomes – a native but insoluble preparation which precludes biochemical access to both sides of the channel. Furthermore, most ion channels are not stable in detergents and determination of the optimal solubility conditions is expensive and time-consuming. NABBs serve as a novel system to circumvent these issues. NABBs are small discoidal lipid patches whose rim is stabilized by an amphipathic protein, called apolipoprotein A-I (apo A-I), derived from zebrafish. We use NABBs with incorporated channels to probe the dynamic aspects of ion channel modulation in a native lipid environment.