Abstract
CLC anion/proton exchangers control the pH and [Cl−] of the endolysosomal system that is essential for cellular nutrient uptake. Here, we use heterologous expression and whole‐cell electrophysiology to investigate the regulation of the CLC isoforms ClC‐3, ClC‐4, and ClC‐5 by the adenylic system components ATP, ADP, and AMP. Our results show that cytosolic ATP and ADP but not AMP and Mg2+‐free ADP enhance CLC ion transport. Biophysical analysis reveals that adenine nucleotides alter the ratio between CLC ion transport and CLC gating charge and shift the CLC voltage‐dependent activation. The latter effect is suppressed by blocking the intracellular entrance of the proton transport pathway. We suggest, therefore, that adenine nucleotides regulate the internal proton delivery into the CLC transporter machinery and alter the probability of CLC transporters to undergo silent non‐transporting cycles. Our findings suggest that the CBS domains in mammalian CLC transporters serve as energy sensors that regulate vesicular Cl−/H+ exchange by detecting changes in the cytosolic ATP/ADP/AMP equilibrium. Such sensing mechanism links the endolysosomal activity to the cellular metabolic state.
Highlights
CLC anion channels and anion/proton exchangers fulfill indispensable functions in nerve and muscle excitation, endocytosis, exocytosis, and lysosomal function [1,2]
A good example are the proteins of the AMPK family (AMP-activated protein kinases) in which the CBS domains serve as energy sensors detecting changes in the ATP/ADP/AMP levels
We transfected HEK293T cells and used whole-cell electrophysiology to investigate the functional effects of adenine nucleotides on three CLC anion/proton exchanger isoforms, the human ClC-4 and ClC-5, and the membrane-localized mouse ClC-3 splice variant ClC-3c
Summary
CLC anion channels and anion/proton exchangers fulfill indispensable functions in nerve and muscle excitation, endocytosis, exocytosis, and lysosomal function [1,2]. CLC proteins assemble as dimers of two identical subunits with parallel orientation and separate ion transport pathways. CBS domains are adenine-nucleotide-binding structures found in many unrelated protein families; their physiological function has been established by association with various human hereditary diseases [8,9,10,11]. A good example are the proteins of the AMPK family (AMP-activated protein kinases) in which the CBS domains serve as energy sensors detecting changes in the ATP/ADP/AMP levels (see [12] for a review). Only the structure of the ClC-5 C-terminus is described revealing an adenine nucleotide binding pocket formed between the two cytoplasmic CBS domains and occupied either by ADP or ATP
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