Trafficking of the Ca2+-activated K+Channel, hIK1, Is Dependent upon a C-terminal Leucine Zipper

Abstract

We demonstrate that the C-terminal truncation of hIK1 results in a loss of functional channels. This could be caused by either (i) a failure of the channel to traffic to the plasma membrane or (ii) the expression of nonfunctional channels. To delineate among these possibilities, a hemagglutinin epitope was inserted into the extracellular loop between transmembrane domains S3 and S4. Surface expression and channel function were measured by immunofluorescence, cell surface immunoprecipitation, and whole-cell patch clamp techniques. Although deletion of the last 14 amino acids of hIK1 (L414STOP) had no effect on plasma membrane expression and function, deletion of the last 26 amino acids (K402STOP) resulted in a complete loss of membrane expression. Mutation of the leucine heptad repeat ending at Leu 406 (L399A/L406A) completely abrogated membrane localization. Additional mutations within the heptad repeat (L385A/L392A, L392A/L406A) or of the a positions (I396A/L403A) resulted in a near-complete loss of membrane-localized channel. In contrast, mutating individual leucines did not compromise channel trafficking or function. Both membrane localization and function of L399A/L406A could be partially restored by incubation at 27°C. Co-immunoprecipitation studies demonstrated that leucine zipper mutations do not compromise multimer formation. In contrast, we demonstrated that the leucine zipper region of hIK1 is capable of co-assembly and that this is dependent upon an intact leucine zipper. Finally, this leucine zipper is conserved in another member of the gene family, SK3. However, mutation of the leucine zipper in SK3 had no effect on plasma membrane localization or function. In conclusion, we demonstrate that the C-terminal leucine zipper is critical to facilitate correct folding and plasma membrane trafficking of hIK1, whereas this function is not conserved in other gene family members.

Role of the NH2 Terminus in the Assembly and Trafficking of the Intermediate Conductance Ca2+-activated K+ Channel hIK1

“…Finally nuclei were labeled with Hoechst 33258 (Sigma). This approach allowed us to detect both cell surface and intracellular HA-hIK1 in the same cells as described previously (20). Cells were then subjected to laser confocal microscopy using a Leica TCSNT 3 laser 4 PMT system.…”

“…HA (YPYDVPDYA) and Myc (EQKLISEEDL) epitope tags were inserted either into the second extracellular loop between Gly 132 and Ala 133 or at the COOH terminus, respectively, as described previously (20). All mutations in the HA-hIK1 channel were produced using the QuikChange TM site-directed mutagenesis strategy (Stratagene).…”

“…20 g of total protein was run on the gel unless otherwise noted in the text. Immunoblotting was then carried out as described previously (20). In experiments where band densities are plotted, the band intensities were determined using the UN-SCAN-IT gel TM automated digitizing system (Silk Scientific Corp., Orem, UT).…”

“…Function of hIK1-We previously demonstrated that the correct trafficking of hIK1 to the cell surface was dependent upon a COOH-terminal leucine zipper (20). The 26-amino acid cytoplasmic NH 2 terminus of hIK1 also contains two potential overlapping leucine zippers, resulting in the generation of a dileucine motif (see Fig.…”

“…The hIK1 channel lacks a characteristic T1 assembly domain, conserved in many of the voltage-activated channels (19), and is therefore likely to possess a novel domain that aids in channel assembly and trafficking. Our laboratory recently defined a role for the COOH terminus of hIK1 in the trafficking of the channel to the cell membrane (20). We reported that a leucine zipper located in this region of the channel was crucial for channel folding and membrane trafficking but was not essential for channel assembly.…”