Jeremy D. Bushman - US grants

DESCRIPTION (provided by applicant): The goal of this research is to establish a general mechanism of ion channel gating for potassium channels in pursuit of a functional model for use in therapeutic development. The ATP-sensitive potassium (KATP) channel regulates ion flow across the plasma membrane based on interactions with relative levels of intracellular nucleotides. It is a principal player in many organ systems for controlling cell excitability;genetic mutations identified in patients have correlated channel dysfunction with disease;and several channel ligands have been developed to treat KATP-related disorders. Similar to other K+ channels and ion channels in general, KATP channels and other members of the inward rectifier (Kir) channel family have an intrinsic mechanism for restricting ion passage through changes in their conformation. The stochastic opening or closing of ion channels - termed "gating" - and the conformational changes in the protein complex that lead to gating are fundamental to understanding their role in physiology. This work will examine which structural elements of the KATP channel in the pore are involved in gating by utilizing known disease-causing mutations and selective mutagenesis to probe function with electrophysiology. Current structural and functional analysis of Kir channels suggest the M2 helices of Kir6.2, which line the pore of the channel, bend at a glycine position (the hinge) and splay apart at the intracellular end to open the pore at the helix bundle crossing. A preliminary examination of the upper glycine (G156 near selectivity filter) in the M2 helices indicates a necessary structural role for this residue during gating. Aim 1 characterized a clinical mutation G156R that produced complete loss of channel activity at the surface but was rescued with a second pore mutation, and further experiments elucidate implications of reconstituting gating function. The next two Aims examine the two transmembrane gates - selectivity filter (Aim 2) and helix bundle crossing (Aim 3) - and their dependence on the G156 residue to determine whether each domain works individually or cooperatively to gate the pore. The Aims will also enable me to draw conclusions about the validity of the hinge hypothesis in KATP channels. ATP-sensitive potassium channels selectively conduct potassium ions through changes in their structural conformation. Understanding what elements of protein structure are important for conformational transitions between one functional state and another will enable drug design to target a specific state of the channel to control its function. Selective drugs will improve alleviation of many disorders caused or ameliorated by the KATP channel.

DESCRIPTION (provided by applicant): The taste system plays a major role in informing organisms on the choice of diet and, consequently, understanding the functioning of this system may lead to the development of treatments for health conditions that are subject to dietary influences including obesity, diabetes, and heart disease. Five basic taste qualities - sweet, salty, bitter, umami, and sour - are encoded by peripheral cells within taste buds of the tongue and encoded into distinct qualities. For many of these, the receptors have been identified and shown to be expressed in subsets of cells within the taste bud. The receptor for sour is currently unknown, but sour cells are faithfully marked by expression of the Transient Receptor Potential (TRP) ion channel Pkd2l1. Intriguingly, accumulating evidence suggests that sour cells have the capacity to transmit or modulate sensations other than sour. For example, the aversive response to high salt is mediated, in part, by cells that express Pkd2l1. In addition, activation o G protein-coupled receptors (GPCRs) and ion channels that do not function as sour receptors can cause release of neurotransmitters from sour cells. Detailed knowledge of the molecular constituents of sour cells will help establish signaling pathways involved in sour cell activity. I preliminary work, the transcriptome of Pkd2l1-expressing sour cells was profiled to identify putative receptors and signaling intermediaries involved in sour and potentially other taste qualities. Results showed the unique expression of two previously unidentified TRP channels in sour taste cells. TRP channels are expressed in numerous sensory cell types and can function as receptors for extracellular stimuli and as sensors of the intracellular environment. The proposed work will test the following HYPOTHESIS: Two previously unidentified TRP ion channels are downstream effectors of intracellular signaling cascades that stimulate sour cell activity. Using computational approaches, molecular and cellular methods, and electrophysiology, this proposal presents experiments aimed at substantiating the roles of these two TRP channels in taste signaling. By furthering our understanding of the molecular physiology of the taste system, this work may lead to the development of strategies to combat human diseases that result from poor dietary choices.