Project Summary The pro-arrhythmic long QT syndrome (LQTS) is commonly caused by drugs or mutations that decrease the amplitude of the rapidly activating delayed rectifier K+ current in the heart (IKr). Macroscopic IKr is a direct function of the number of Kv11.1 channels in the cell surface. This in turn, depends on the balance between channel insertion, recycling and degradation. About 90% of LQTS-linked missense mutations in KCNH2 decrease Kv11.1 channel number in the cell surface by disrupting channel trafficking. The trafficking for many of these mutants is increased by culturing cells in drugs that block Kv11.1 current (IKv11.1). We propose the innovative hypothesis that some of these mutations increase the activity of Kv11.1 channel in early endosomes (EE), recycling endosomes (RE) and/or the trans-Golgi network (TGN). The increase in channel opening in these organelles alters the organelle membrane potential (ψ), pH and K+ levels. The changes in the organelle electrochemical gradients alter the conformational of Kv11.1 channels that prevent their onward trafficking and/or promote degradation. Drugs that block IKv11.1 prevent mutant channel openings, prevent organelles changes in ψ, pH and K+ levels, and improve onward trafficking/decrease degradation. To test this hypothesis, we will develop a method to assay Kv11.1 channel opening in selected, relevant organelles for membrane insertion, recycling, and degradation. We can already measure ψ in EEs, REs and the TGN using a DNA-based reporter called Voltair, developed by Krishnan. Now, we will use a DNA-based reporter for K+ we have recently developed, called pHlicKer, to simultaneously quantitate lumenal pH and [K+] in these organelles. We will engineer variants of pHlicKer that localize specifically in EEs, REs, or the TGN in live cells. We will then use Voltair and pHlicKer to explicitly determine how mutant Kv11.1 channels that increase channel opening in these organelles impacts ψ, lumenal pH and lumenal [K+] levels. We expect that mutations that increase the opening of Kv11.1 channels will decrease ψ and increase lumenal [K+]. The changes in the electrochemical gradients in EEs, REs, or the TGN will prevent the onward trafficking/promote the degradation of Kv11.1 channels to the cell surface. We expect incubating cells expressing mutant Kv11.1 channels in drugs that block IKv11.1 will prevent the changes in the electrochemical gradients of EEs, REs, and TGN to improve mutant Kv11.1 channel trafficking increase functional half-life. The development of the first-generation prototypes to measure electrochemical gradients in organelles will allow us to quantify how channel dynamics change as they traffic to the plasma membrane. This will be a critical step to develop new molecules that can selectively target intracellular channels intracellular to impact their expression and/or degradation. Our research would also lead to the first practical method to map organellar K+ and potentially a...