Mechanisms of Ion Channels and Enzymatic Membrane Proteins

PROJECT SUMMARY The objectives of this research program are to understand the structural, functional, and molecular mechanisms of two classes of integral membrane proteins in eukaryotes: ion channels and enzymes that catalyze chemical reactions within lipid membranes. For ion channels, we aim to discover the mechanisms by which the channels conduct ions across cellular membranes, achieve ion selectivity, and are gated. Regarding membrane enzymes, the salient questions we will address include how both water-soluble and lipophilic substrates access membrane-embedded active sites, what underlies chemical mechanisms of catalysis, what conformational changes occur during the reaction cycles, and what constraints the lipid membrane places on these processes. We combine approaches to determine three-dimensional structures (X-ray crystallography and cryo-electron microscopy) with functional analyses (e.g. electrophysiology, enzymology, and biochemistry) to pursue holistic mechanistic understandings of these complex molecular machines. The ion channels under study include the mitochondrial calcium uniporter, the bestrophin (BEST) family of calcium-activated chloride channels, and two-pore domain potassium (K2P) channels. The mitochondrial calcium uniporter is a highly regulated multi-subunit ion channel complex. It is the primary conduit for mitochondrial calcium entry and thereby regulates ATP synthesis and other processes. Our efforts are aimed to discover the channel’s modes of ion selectivity and gating, and to investigate functions of its regulatory subunits. BEST channels form anion-selective pores that are regulated by intracellular calcium, phosphorylation, and changes in cell volume. Mutations in BEST channels cause retinal degenerative diseases. Our efforts are geared to understand the gating and selectivity properties of the channels, with particular attention to: differences among human BEST1-4 channels, interactions with binding partners (e.g. lipids and other proteins), and the possibility that the channels conduct neurotransmitters. K2P channels establish the resting potential of cells and thereby regulate immune responses and neuronal firing. We aim to determine the structures and mechanisms of the channels, with emphasis on regulation by cellular binding partners. Regarding our studies of integral membrane enzymes, current focuses are the enzymes ICMT and RCE1, which catalyze posttranslational modifications of RAS and other CAAX proteins, and the enzymes HHAT and GOAT, which attach acyl groups onto the signaling molecules Hedgehog and ghrelin. We aim to determine atomic structures of these enzymes with substrates, substrate analogs, and products that represent snapshots of their reaction coordinates – and to combine these efforts with experiments that address function. The studies will reveal principles of ion channel and enzyme function, thereby making substantial contributions to the understandings of the physiological processes that these membrane proteins control.