K+ is concentrated in all cells and essential for many physiological processes. In bacteria, fungi, plants and protists, membrane proteins from the Superfamily of K+ Transporters, or SKT proteins, mediate homeostasis of K+. The focus of the current proposal is the SKT family member TrkH, which, together with its closely related homologs TrkG and KtrB, forms the largest sub-family of SKT proteins. TrkH/TrkG/KtrB proteins are required for cell growth and are potential targets for antibiotics. The long-term goal of the project is to understand mechanisms of operation for the SKT proteins. Previously, my lab obtained structures of TrkH from Vibrio parahaemolyticus in isolation and in complex with TrkA, a cytosolic protein comprising two Regulate- Conductance-of-K+, or RCK domains. We also measured the first single-channel currents from the TrkH-TrkA complex, and demonstrated that nucleotides binding to TrkA regulate the function of the channel. Based on these exciting results, I propose the following two Specific Aims: to unravel the mechanism of TrkA-controlled gating of TrkH; and to examine the structural basis of ion selectivity and conduction in TrkH. We will also assess how perturbations to TrkH gating, ion selectivity and conduction affect its abilit to rescue E. coli in low K+ concentrations. Combined, results from these experiments will provide new knowledge of structural underpinnings of ion permeation and gating in TrkH, and will eventually lead us to a better understanding of basic mechanisms of SKT proteins.
Aim 1. To investigate the mechanism of gating in the TrkH-TrkA complex.
Aim 2. To examine the mechanism of ion selectivity and permeation in TrkH.
All living cells accumulate potassium ions, and a high intracellular potassium ion concentration is essential in physiology. In bacteria, yeast, and plants, potassium ion uptake is achieved by a superfamily of potassium ion transporters, the SKT proteins, which has more than 6000 members identified to date. Since the SKT family of proteins is not found in humans or other animals, these proteins are potential targets for antibiotics or fungicides. Work proposed in this application will lead us to a better understanding of basic mechanisms of how this large and diverse family selects and conducts ions, and how ion conduction is regulated by intracellular ATP and ADP concentrations; this knowledge will provide a solid foundation for developing small- molecule inhibitors specific to SKT proteins from major pathogens, which could be used as lead compounds for drugs targeting these proteins.
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