Abstract
Many plasma membrane Cl- channels have been cloned, including the cystic fibrosis transmembrane conductance regulator and several members of the voltage-gated ClC family. In contrast, very little is known about the molecular identity of intracellular Cl- channels. We used a polymerase chain reaction-based approach to identify candidate genes in mammalian brain and cloned the cDNA corresponding to rat brain p64H1. This encoded a microsomal membrane protein of predicted Mr 28,635 homologous to the putative intracellular bovine kidney Cl- channel p64. In situ mRNA hybridization histochemistry showed marked expression in hippocampus and cerebellum, and in vitro expression revealed a large cytoplasmic domain, one membrane-spanning segment, and a small nonglycosylated N-terminal luminal domain. The predicted protein contained consensus phosphorylation sites for protein kinase C and protein kinase A, and protein kinase C-mediated phosphorylation increased the Mr of p64H1 to approximately 43,000, characteristic of the native protein in Western blots. Recombinant p64H1 was immunolocalized to the endoplasmic reticulum of human embryonic kidney 293 and HT-4 cells, and incorporation of human embryonic kidney 293 endoplasmic reticulum vesicles into planar lipid bilayers gave rise to intermediate conductance, outwardly rectifying anion channels. Although p64H1 is the first intracellular Cl- channel component or regulator to be identified in brain, Northern blotting revealed transcripts in many other rat tissues. This suggests that p64H1 may contribute widely to intracellular Cl- transport.
Highlights
Understand the molecular basis of intracellular ion transport has revealed the molecular identities of several intracellular cation channels, intracellular ClϪ channels are much less well characterized
Reconstitution of partially purified p64 into planar lipid bilayers revealed anion channel activity [16], and incorporation into proteoliposomes conferred a 36Cl permeability that was abolished by anti-p64 antibodies [17]. p64 cDNA was cloned [17] and expressed in Xenopus oocytes, where the recombinant protein was localized to intracellular membranes
In an attempt to identify p64-related intracellular ClϪ channel proteins in mammalian brain, we used a PCR-based approach to clone and functionally express a rat brain homologue of p64, p64H1. This is the first putative intracellular ClϪ channel component to be identified in brain, and we suggest that p64- and p64H1-related genes encode a family of proteins associated with intracellular ClϪ transport
Summary
Understand the molecular basis of intracellular ion transport has revealed the molecular identities of several intracellular cation channels, intracellular ClϪ channels are much less well characterized. P64 cDNA was cloned [17] and expressed in Xenopus oocytes, where the recombinant protein was localized to intracellular membranes.
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