The Molecular Physiology and Biophysics Unit (MPBU) studies the molecular structure and function of voltage-gated potassium and calcium channel proteins using biochemical, molecular biological and biophysical methods. Voltage-gated potassium and calcium channels are homologous integral membrane proteins that are important for the generation and processing of electrical signals in the nervous system, regulation of heart contraction and the secretion of hormones that regulate blood sugar. In one series of projects we are using hanatoxin (a protein toxin we first isolated from tarantula venom) as both a functional and structural probe to investigate the molecular basis of voltage-dependent gating (opening and closing of the ion passageway) in the superfamily of voltage-gated ion channels. Our primary focus is on voltage-gated potassium channels where hanatoxin binds to four identical receptors on each channel (a homotetrameric protein complex) and alters the energetics of gating. We discovered that hanatoxin can also recognize its receptor on other voltage-gated channels (e.g. calcium channels), thus defining a voltage-sensing domain that has a highly conserved three-dimensional structure in voltage- gated ion channels. We have begun to identify which channel residues form the hanatoxin receptor, and thus the voltage- sensing domain, and determine their localization within the channel. Our experiments suggest that the hanatoxin receptors lie at least 25 angstroms from the central pore axis on the surface of voltage-gated potassium channels. Another aspect of the hanatoxin project is to use the toxin to better understand the conformational changes that occur as voltage-gated channels gate. We discovered that hanatoxin inhibits gating currents, small capacitive currents arising from the initial conformational changes involved in channel gating. Hanatoxin is the first reagent to have such profound effects on gating currents and will be a useful tool to understand what gating currents can tell us about the conformational changes involved in voltage-dependent gating. We are continuing to search for new toxins that target channels whose physiological roles remain to be identified. T-type calcium channels have the unique biophysical properties of activating near the resting membrane potential of many cells and inactivating rapidly. In neurons and cardiac muscle, T-type calcium channels are thought to be involved in pacemaking, while in vascular smooth muscle they may play an important role in setting vascular tone. This year we isolated and have begun to characterize a toxin from scorpion venom that selectively targets the T-type calcium channel. This toxin will be a useful pharmacological probe to characterize the molecular composition of T-type calcium channels and delineate their involvement in specific physiological processes.
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