Cloning and Functional Expression of Rat CLC-5, a Chloride Channel Related to Kidney Disease

Abstract

We have cloned a novel member of the CLC chloride channel family from rat brain, rCLC-5. The cDNA predicts a 83-kDa protein belonging to the branch including CLC-3 and CLC-4, with which it shares ϳ80% identity. Expression of rCLC-5 in Xenopus oocytes elicits novel anion currents. They are strongly outwardly rectifying and have a conductivity sequence of NO 3Although CLC-5 has consensus sites for phosphorylation by protein kinase A, raising the intracellular cAMP concentration had no effect on these currents. Currents were also unchanged when rCLC-5 was coexpressed with rCLC-3 and rCLC-4, either singly or in combination. rCLC-5 is expressed predominantly in kidney and also in brain, lung, and liver. Along the nephron, rCLC-5 message is detectable in all tubule segments investigated, but expression in the glomerulus and the S2 segment of the proximal tubule is low.

Sorting Motifs of the Endosomal/Lysosomal CLC Chloride Transporters

“…2A). However, mutating Leu 47 and Ile 48 to alanines did not significantly reduce the binding of APs and clathrin to ClC-5-NT, whereas, surprisingly, mutating Tyr 14 to Ala strongly reduced these interactions (Fig. 2C).…”

“…Because the crystal structure of the prokaryotic EcClC-1 protein suggests that the third motif might extend into the first membrane-spanning helix B (43), we focused on the first two motifs. Mutating Tyr 48 to Ala strongly reduced AP-2 and AP-3 binding, with a weaker effect seen with the Y61A mutant (Fig. 2D).…”

“…1A). We, therefore, initially tested another sequence in ClC-5-NT (ESTWALI 48 ) that almost matches the (DE)XXX-L(LI) consensus sequence ( Fig. 2A).…”

A Two-Holed Story: Structural Secrets About ClC Proteins Become Unraveled?

“…The organelle channels ClC-3, ClC-4, and ClC-5 can be classified as a subfamily characterized by a strong outward rectification when expressed in Xenopus oocytes or mammalian cells (16,23,43). ClC-6 and ClC-7 (6) form the third branch of the ClC family.…”

Expression and targeting to the plasma membrane of xClC-K, a chloride channel specifically expressed in distinct tubule segments of Xenopus laevis kidney

“…Whereas the mammalian kidney develops into a typical metanephros, the batrachian kidney stops its development at the mesonephros stage [33]. XClC-K is the second Cl − channel predominantly expressed in kidney that has been cloned from Xenopus after xClC-5 [34], the homologue of mammalian ClC-5 channels [35]. Because both display properties very similar to their respective mammalian counterparts, this suggests that Cl − transport relies on a common set of ClC channels acting in a similar fashion in both primitive and more evolved kidneys.…”