Abstract
pICln is a protein that induces an outwardly rectifying, nucleotide-sensitive chloride current (ICln) when expressed in Xenopus oocytes, but its precise function (plasma-membrane anion channel versus cytosolic regulator of a channel) remains controversial. We now report that a chloride current identical to ICln is induced when Xenopus oocytes are injected with human ClC-6 RNA. Indeed, both the pICln and the ClC-6 induced current are outwardly rectifying, they inactivate slowly at positive potentials and have an anion permeability sequence NO3- > I- > Br- > Cl- > gluconate. Cyclamate, NPPB, and extracellular cAMP block the induced currents. The success rate of current expression is significantly increased when the injected Xenopus oocytes are incubated at a higher temperature (24 or 37 degrees C) prior to the analysis. In addition, the ICln current was detected in 6.2% of noninjected control Xenopus oocytes. We therefore conclude that the ICln current in Xenopus oocytes corresponds to an endogenous conductance that can be activated by expression of structurally unrelated proteins. Furthermore, functional, biochemical, and morphological observations did not support the notion that pICln resides in the plasma membrane either permanently or transiently after cell swelling. Thus, it is unlikely that pICln forms the channel that is responsible for the ICln current in Xenopus oocytes.
Highlights
PICln is a 26-kDa acidic protein that is ubiquitously expressed in various mammalian cell lines and tissues and in Xenopus oocytes [1,2,3]
Expression of the pICln-associated Chloride Current in Xenopus Oocytes Is Temperature-dependent—It has previously been reported that expression of mammalian pICln in Xenopus oocytes resulted in an outwardly rectifying chloride current (ICln) that slowly inactivated at positive potentials and that could be blocked by extracellular nucleotides [4, 16, 17]
Only Xenopus oocytes that had been injected with human pICln and that had been subjected to a temperature shift displayed ICln-type currents
Summary
PCR, reverse transcription-PCR, and Vector Construction—A human cDNA clone for pICln (accession number X91788; see Ref. 1) was PCR-mutagenized by replacing the 5Ј-untranslated region with an EcoRI/HindIII/BamHI linker. First we amplified by reverse transcription-PCR the 5Ј-end of human ClC-6 (nucleotides 202–1229 of the published open reading frame; see Ref. 12). Nucleotides 1–201 of the open reading frame were amplified by reverse transcription-PCR from human K562 RNA and inserted upstream of the ClC-6 sequence via a NcoI site. This created a pBluescript vector containing the complete ClC-6 open. Cells were permeabilized with 0.5% Triton X-100 and blocked with 10% goat serum in PBS for 1 h They were incubated with the affinity-purified polyclonal anti-pICln antibodies for 36 h at 4 °C and thereafter with fluorescein isothiocyanate-conjugated antirabbit IgG (Sigma) at room temperature for 1 h. Cell Volume Measurements—EA.hy926 cells were grown on glass coverslips, and cell height was monitored as described by Van Driessche et al [15]
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