Molecular Identity and Physiological Function of Novel Chloride Channels

PROJECT SUMMARY/ABSTRACT Chloride is the most abundant free anion in animal cells. Chloride channels play a wide range of functions including cell volume regulation, fluid secretion, regulation of excitability, and acidification of intracellular organelles. Their physiological role is impressively illustrated by many genetic diseases involving chloride dysregulation, such as cystic fibrosis, myotonia, and epilepsy. However, despite recent progress, chloride channels have long suffered as poor cousins in the aristocratic family of ion channels. For decades, the field has been dominated by sodium, potassium and calcium channels. Indeed, there are still many electrophysiologically well-characterized chloride channels without molecular identity. This gap makes it impossible to elucidate their precise function and how their dysfunction leads to disease. In the previous R35 MIRA ESI funding period, we performed an unbiased RNAi screen and identified PAC, a novel membrane protein with no sequence similarity to other ion channels, as the long sought-after acid or proton-activated chloride (PAC) channel. By mediating chloride influx and subsequent cell swelling, PAC currents have been implicated in acid-induced cell injury. We generated PAC knockout mice and demonstrated that PAC plays a key role in acid-induced cell death in vitro and ischemic brain injury in vivo. Thus, PAC is a potential drug target for stroke and other acidosis-associated diseases. By combining mutagenesis, patch-clamp recording, and cryo-EM, we revealed for the first time the trimeric assembly, ion conducting pathway, the basis of anion selectivity, pH-dependent conformational change, and pH-sensing mechanism of this new channel. Discovery of a novel channel represents a breakthrough that opens up a new field. In the next 5 years, we will focus on the diverse regulatory mechanisms of the PAC channel and its surprising physiological function in vesicular acidification that we have recently discovered. The long-term goal of this MIRA program is to apply a multi-disciplinary approach including high-throughput functional genomics, patch-clamp electrophysiology, structural biology, imaging, and mouse genetics to the underexplored area of chloride channel biology.