Fluctuations in Ionic Current Through Membrane Channels

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DESCRIPTION (provided by applicant): About 30% of the human genome codes for membrane proteins, but membrane protein structures account for far fewer than 1 % of the entries in the Protein Data Bank. Of particular interest to neuroscience would be structures of ion channels and membrane receptors, even at low resolution but in multiple functional states so that the molecular motions that underlie their actions are visualized. Toward this goal we are working on a general and flexible method for the imaging of membrane proteins, in a membrane environment, using electron microscopy of cryogenically-cooled specimens (cryo-EM) and computer image processing. The application of this method will first be to the IPS receptor, an intracellular calcium release channel that is responsible for calcium signaling in brain and in many other cell types. We plan to observe this channel's structure in its closed and open states, in membrane vesicles. The large-conductance, calcium and voltage-activated potassium channel (BK channel) has roles ranging from the provision of local feedback in presynaptic nerve terminals to the control of blood pressure. Its characteristics also make it particularly suited to structural studies of the S4 voltage-sensor of voltage gated ion channels. Our goal is to observe the structure of its voltage sensor in both the activated and deactivated states, while the channel is in a membrane environment. To complement the structural work on the BK channel we seek to measure the time course of its conformational changes using fluorescence techniques. Making use of a library of fluorescent BK channel fusion proteins, we will employ fluorescence energy transfer to monitor changes in inter-domain distances in BK channels in a patch-clamped membrane.