Abstract
ClC-3 is a member of the CLC family of anion channels and transporters, for which multiple functional properties and subcellular localizations have been reported. Since alternative splicing often results in proteins with diverse properties, we investigated to what extent alternative splicing might influence subcellular targeting and function of ClC-3. We identified three alternatively spliced ClC-3 isoforms, ClC-3a, ClC-3b, and ClC-3c, in mouse brain, with ClC-3c being the predominant splice variant. Whereas ClC-3a and ClC-3b are present in late endosomes/lysosomes, ClC-3c is targeted to recycling endosomes via a novel N-terminal isoleucine-proline (IP) motif. Surface membrane insertion of a fraction of ClC-3c transporters permitted electrophysiological characterization of this splice variant through whole-cell patch clamping on transfected mammalian cells. In contrast, neutralization of the N-terminal dileucine-like motifs was required for functional analysis of ClC-3a and ClC-3b. Heterologous expression of ClC-3a or ClC-3b carrying mutations in N-terminal dileucine motifs as well as WTClC-3c in HEK293T cells resulted in outwardly rectifying Cl(-) currents with significant capacitive current components. We conclude that alternative splicing of Clcn3 results in proteins with different subcellular localizations, but leaves the transport function of the proteins unaffected.
Highlights
Alternative splicing can result in proteins with distinct subcellular distributions and functions
Since alternative splicing often results in proteins with diverse properties, we investigated to what extent alternative splicing might influence subcellular targeting and function of ClC-3
We amplified ClC-3 splice variant from different mouse tissues by RT-PCR (Fig. 1A) and demonstrated that only three splice variants are expressed in the brain, the olfactory bulb and the spinal cord, ClC-3a, ClC-3b, and ClC-3c, with ClC-3b and ClC-3c being the predominant ClC-3 splice variants (Fig. 1C and Fig. 2)
Summary
Alternative splicing can result in proteins with distinct subcellular distributions and functions. Results: Three ClC-3 splice variants are expressed in the mammalian brain with different subcellular localizations, but identical transport properties. Changes in synaptic transmission in these animals suggest that ClC-3 is present in synaptic vesicles and contributes to the regulation of neurotransmitter accumulation and release from the presynaptic nerve terminal [2, 4, 5]. A potential reason for such functional differences between native and heterologously expressed proteins might be the existence of alternatively spliced ClC-3 variants with distinct subcellular localizations and transport functions. We found three splice variants that differ in the N-terminal domain and exhibit identical transport function, but different subcellular distributions
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