The Amino Terminus Controls Sorting and Voltage Dependence of CLC 3

Abstract

ClC-3 is a member of the CLC family of anion channels and anion-proton exchangers. So far, no agreement has been reached its biophysical properties and its subcellular localization. Some groups reported ClC-3 being an intracellular transporter, whereas other experimental evidence supports anion channel function and plasmalemmal localization. Since alternative splicing might result in distinct ClC-3 proteins with diverse functions we decided to compare biophysical properties and subcellular localization of known ClC-3 splice variants, ClC-3A and ClC-3B, and to compare them with the well characterized short isoform ClC-3short (ACS Chem Neurosci 4, 994-1003). Both isoforms target exclusively to intracellular compartments, ClC-3A to the endosomal/lysosomal system and ClC-3B to Golgi apparatus. ClC-3short contains a dileucine-like motif in its amino terminus, and we here identified two additional dileucine-like motifs in this region of ClC-3A/B. These motifs interact with clathrin, and their disruption permit surface membrane insertion and electrophysiological analysis of ClC-3A/B. Heterologous expression of ClC-3A/B26/41/71 in HEK293T cells results in outwardly rectifying Cl- currents with amplitudes up to 2 nA. Similar to the short isoform, ClC-3A26/41/71 and ClC-3B26/41/71 exhibit significant capacitive current components indicating that there is a high probability of non-complete transport cycles under our conditions. For ClC-3A/B26/41/71 the ratio of transport currents and gating charge is significantly lower than for the short one. Moreover, the voltage dependence of charge movement was shifted to more positive potentials compared to the short isoform. The kinetic of the gating charge movements was faster for ClC-3A26/41/71 than the ClC-3B26/41/71. Our results show that all known ClC-3 splice variants are transporters, with significant capacitive currents and pronounced outward rectification. N-terminal alternative splicing does not only affect trafficking, but also influences the voltage dependence of ClC-3, permitting adjustment of transport functions to distinct physiological roles.