Mechanisms that Alter Potassium Channel Trafficking in Arrhythmias

Specific Aims: The number of K+ channel proteins at the cell surface reflect the intersection between three trafficking pathways: channel insertion in the plasma membrane, channel recycling and channel degradation. This application tests a fundamentally new hypothesis: that the opening of K+ channels within an organelle regulates its trafficking and abundance at the cell surface. We will test this by studying the trafficking of the KCNH2-encoded Kv11.1 channels. Kv11.1 channels conduct the rapidly activating delayed rectifier K+ current (IKr) in the heart. We chose to study Kv11.1 channels because mutations in KCNH2 that disrupt Kv11.1 channel trafficking are a common cause of the deadly long QT syndrome (LQTS). We have studied >100 different LQTS-linked KCNH2 missense mutations and ~90% of them are trafficking-deficient. Drugs that bind to Kv11.1 channels and block Kv11.1 current (IKv11.1) can improve the trafficking of mutant Kv11.1 channel at the cell surface membrane. This suggests that some LQTS-linked KCNH2 mutations might disrupt the trafficking of Kv11.1 channels by causing aberrant channel openings in organelles. We recently developed Voltair and discovered that the trans-Golgi (TGN), Recycling Endosomes (RE), and Early Endosomes (EE) have significant membrane potentials (ψ). Their large ψ and acidic lumenal pH suggest that most Kv11.1 channels are normally closed in organelles. We propose channel opening in organelles regulates channel trafficking by altering the electrochemical gradient across the organelle membrane. We can now test this hypothesis because we developed the tools to measure the electrochemical gradients in the TGN, RE, and EE in live cells. pHlicKer is the first pH-correctable K+ reporter to measure the lumenal [K+] in these organelles. Voltair and pHlicKer will allow us to measure K+ channel activity in these organelle because increasing Kv11.1 channel openings is expected to decrease organelle ψ and increase lumenal [K+]. We will determine whether trafficking-deficient mutant Kv11.1 channels are prone to channel opening in these ionic conditions experienced in TGN, RE, or EE (Aim 1). We will then determine whether trafficking-deficient Kv11.1 mutations that increase Kv11.1 channel opening disrupt electrochemical gradients in the TGN, RE, or EE in live cells (Aim 2). For the first time, these studies would implicate changes in the organellar electrochemical gradients as a new cellular mechanism for the regulation of channel trafficking to and from the cell surface membrane. Aim 1: Test Kv11.1 channel mutants for aberrant activity in electrochemical gradients equivalent to those in organelles. Premise: Kv11.1 channel activity depends on ψ and pH gradients. Hypothesis: The large ψ and low pH in the lumen of organelles keeps wild type (WT) Kv11.1 channels in non- conducting closed states. Trafficking-defective Kv11.1 mutations increase channel activity (openings) compared to WT-Kv11.1 channels in the ψ and low pH experienced in organelles. Approach: We will use whole-cell voltage-clamp to measure Kv11.1 currents (IKv11.1) in cells expressing WT- or mutant channels under transmembrane gradients of ψ, pH and K+ that simulate TGN, the EE or the RE organelle environments. Aim 2: Determine how organellar K+, pH and ψ change due to Kv11.1 channel activity in the organelle. Premise: We developed Voltair and pHlicKer which allows us to measure ψ, pH and K+ in the TGN, EE or the RE organelle in live cells. Hypothesis: Cells expressing trafficking-deficient mutations that increase Kv11.1 channel opening will decrease organelle ψ and increase lumenal [K+] compared to cells expressing WT-Kv11.1 channels. Drugs that improve mutant Kv11.1 channel trafficking will normalize mutation-induced changes in organelle ψ and lumenal [K+]. Approach: Using Voltair and pHlicKer we will measure ψ, lumenal pH and K+ cells will be expressing different levels of WT-Kv11.1 channels or trafficking-deficient Kv11.1 channels. We will test whether cells expressing mutant channels will increase heterogeneity in ψ, lumenal pH and [K+] in organelles. We will also determine if drugs that increase trafficking of mutant Kv11.1 channels normalize the mutant-induced changes in the organelle ψ, lumenal pH and [K+] and increase functional half-life of mutant Kv11.1 channels. Impact: Voltair and pHlicKer technology is amenable to cell-based high throughput screens. Their use is expected to accelerate the discovery of selective modulators of K+ channel activity in organelles, and produce new knowledge of mechanisms that modulate the cell-surface abundance of K+ channels to counter conditions like LQTS. Finally, our approach can also be adapted to study other K+ channels and organellar K+ channels at an entirely new level of detail.