Chloride fluxes in organellar membranes

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications & trials

Abstract

This project is using a combination of methods to analyze the ion transport properties of lysosomal membranes with particular focus on their role in generating the acid luminal pH of the organelle. Lysosomes are intracellular organelles that serve in most cells as digestive organelles although in some tissues they are used for other functions. Lysosomes utilize an ATP-driven proton pump to maintain an acidic luminal pH and facilitate their digestive function. Such a pump can only be effective if accompanied by additional ion transport to dissipate the transmembrane voltage built up by the ATPase, a counterion pathway. In the past, we used isolated lysosomes to identify and characterize a Chloride permeability in the lysosomal membrane which has the features required of such a counterion pathway and demonstrated that the chloride is transported by ClC-7, a Cl-/H+ antiporter specifically targeted to the lysosomal membrane. Using optimized optical methods to measure lysosomal pH, we are analyzing the effects of cytoplasmic ionic composition and ClC-7 knockout on these processes. In addition to our work on lysosomes, we have begun to explore the acidification process in other organelles. Finally, we have been collaborating with the Gahl lab in the Genome institute to analyze a mutant form of ClC-7 that they have found in a patient in the Undiagnosed Diseases Program who has a disease pattern completely unlike those found with other ClC-7 mutations. We have made substantial progress in understanding the effects of this mutation, with careful analysis of the effects of the mutation on ClC-7 function, the effects of these changes on pH in lysosomes, and possible approaches to treating this disease, published just before the COVID pandemic. We demonstrated that the mutation causes patient lysosomes to become hyperacidic, and that change in organellar pH leads to a host of disruptions of cellular metabolism. We also showed that we could correct the cellular defects by treating cells with agents known to alkalinize lysosomes. These approaches suggest possibilities for therapy of the disease. We are currently following up on this work to explore the regulation of ClC-7 by intrinsic cellular stimuli as well as the structural mechanism by which the patient mutation perturbs ClC-7 function.