Goldstein Lab Research Interests

The structural basis for potassium channel function

Our work is directed towards understanding the basic molecular mechanisms by which ion channel proteins work. These integral membrane proteins catalyze the selective transfer of ions across membranes and, like enzymes, they show exquisite specificity and tight regulation. As a class, channels play a central role in the transmembrane electrical signals which underlie cellular function in plants and animals. Remarkably, solutions to the most fundamental mechanistic questions about these molecules remain to be discovered. How do they open and close? How can they distinguish a potassium ion from a sodium ion yet maintain turn-over rates of 100 million ions per second? What is the architecture of these extraordinary transmembrane portals? We use a combination of biophysical, molecular biological and protein-biochemical methods to study potassium channels. Our research focuses in five areas.

(1) The structural basis for function of the MinK-related peptides (MiRPs). MinK has 130 amino acids and a single transmembrane domain. It is expressed widely in humans. In the heart it mediates cardiac repolarization by formation of mixed complexes with a pore-forming channel subunit called KvLQT1. Now, we have identified peptides related to MinK that subserve a similar role.  MiRPs are critical to normal function of the heart as their inheritance in mutant form leads to life-threatening arrhythmias).  Yet, our understanding of their function is limited: are they  essential or regulatory? what is the physical architecture of mixed channel complexes? what attributes do they determine? why do mutations cause disease?  We argue that MiRPs contribute directly to formation of the channel pore to define the basic functional characteristics of mixed complexes (e.g., conductance, gating, regulation and pharmacology).

(2) Potassium channel subunits with two P domains. The first member of this family, TOK1, we isolated from the budding yeast Saccharomyces cerevisiae. TOK1 was novel in two respects. First, in contrast to previously known potassium channels that have one pore-forming P region, TOK1 has two. Second, TOK1 was the first cloned example of a new functional type of potassium channel, an 'outward rectifier.' Unlike voltage-gated or inward rectifier channels, an outward rectifier passes outward current in a fashion that is coupled to the equilibrium reversal potential for potassium. Subsquently, we cloned ORK from Drosophila and found it to be the first molecular example of a background (or leak) channel.  As a dizzying array of 2 P domain channels emerges from yeast, flies, mice and humans we seek to understand their role in physiology and to learn how they are similar and how they differ from their 1 P domain predecessors. (3) The role of potassium channels in the heart in health and disease. The molecular basis for ion channel function became accessible through studies of cloned channel genes. Now, it is possible to relate these mechanistic insights to the human heart. We are studying how normal cardiac channels form and function, how disease-producing variants are altered and working to identify channel genes associated with cardiovascular disease. (4) The 3D structure of potassium channels. Here, we take indirect and direct approaches. In the former, we use peptides of known 3D structure as "molecular calipers" to measure distances between channel residues making up their binding sites. Thus, the peptide blocker charybdotoxin (CTX) binds to a site in the pore of Shaker potassium channels and to offer an indirect image of the outer channel pore. We are now studying peptide toxins that interact with 2 P domain channels to explore these unique structures.  Our studies exploiting direct spectroscopic imaging of purified channel proteins employ NMR and X-ray crystallographic methodologies. (5) Ion channel structure and function using yeast molecular genetics. While classical site-directed mutagenesis can teach a great deal when coupled with functional analyses it is dependent on choosing the "correct" site to study. By expressing ion channels in yeast cells that require channel function for survival it has become possible to use random mutagenesis and selective pressure to reveal the mechanistic basis for channel function, regulation and pharmacology.
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