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First published online 16 June 2005
doi: 10.1242/jcs.02423

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Identification of the cyclic-nucleotide-binding domain as a conserved determinant of ion-channel cell-surface localization

Department of Physiology, McGill University, 3655 Promenade Sir William Osler, Montreal, QC H3G 1Y6, Canada

View larger version (48K): [in a new window]   Fig. 1. Primary and secondary structural features of the CNBD. (A) ClustalW multiple sequence alignment of the CNBD of HERG with respect to that of the bacterial transcription factor (CAP), HCN2, KAT1 and ERG3 channels. Regions of secondary structures of CAP (Weber and Steitz, 1987) are indicated at the top and applied to the alignment of HCN2 (Zagotta et al., 2003). White arrows point to LQT2 missense mutations that fall within the putative CNBD of HERG at indicated residues. Black and gray arrows indicate residues of ERG3 and HCN2, respectively, that have been mutated in this study.

View larger version (38K): [in a new window]   Fig. 2. Residue 822 of the putative CNBD is crucial for trafficking of HERG. (A) Cells expressing HERGwt, HERGV822M and HERGV822P mutants were treated with dimethylsulfoxide (control) or proteasomal inhibitors (lactacystin, ALLN, MG132) 1 day after transfection. Cells were lysed and equal amounts of the soluble fraction were separated on SDS-PAGE and subjected to immunoblotting with an anti-HA antibody. (B) Live cells expressing the mutant channel were treated with proteinase K (PK) (+) or untreated (–). PK was blocked with extensive washes and equal amounts of detergent soluble fraction was subjected to immunoblot analysis for HA and CASK (left) and N-cadherin (N-Cad) (right). The arrow (Deg) indicates the product generated by the PK. Quantifications were performed by measuring pixel intensity of the HA and the N-Cad signals before and after PK treatment and normalized to the corresponding CASK signal. (C) Patch clamp analysis of cells co-transfected with CD8 cDNA along with HERGwt and the HERGV822M mutant. Mock transfections were performed with CD8 cDNA. Depolarization steps were imposed on cells decorated with beads.

View larger version (33K): [in a new window]   Fig. 3. LQT2 mutations in the putative CNBD of HERG prevent surface localization caused by ER retention. (A) PK analysis of natural mutations in the putative CNBD of HERG. Membranes were simultaneously probed with anti-HA and anti-CASK antibodies. Arrows indicate the CASK and the mature band of HERG (M) at their respective molecular weights. (B) Quantification of representative experiments described in A (n indicates the number of different experiments used for quantification). Histogram bars show PK insensitivity (black) or significant sensitivity (white, P<0.05). In all cases, the HA signals were normalized to the corresponding CASK signal. For HERGR823K, the pixel intensities of both the immature and the mature bands were considered. (C) Quantification of tail-current density generated in cells expressing HERGwt channel or the indicated mutants. Transfected cells were selected as described and subjected to depolarization steps. Currents were measured at –60 mV and normalized to individual cell capacitance. Black bars indicate mutants with similar activity to mock-transfected cells (Mock).

View larger version (62K): [in a new window]   Fig. 4. Truncation of the CNBD results in ER retention. (A) Schematic representation of HERG with six transmembrane segments (S1-S6). Downward arrow indicate residues of HERG corresponding to the putative CNBD, PAS domain (black arrows) and a previously identified segment involved in channel trafficking (gray arrows). HERG truncated constructs analysed in this study are indicated by the upward arrows. Maturation state corresponding to individual mutants is indicated as X (defective trafficking) or (normal trafficking). (B) Immunoblots of truncation constructs co-expressed with green fluorescent protein (GFP) to compare the transfection efficiency. Transiently transfected cells were lysed and equal amounts of solubilized protein were subjected to SDS-PAGE. The top membrane was probed with an anti-Myc antibody, stripped and reprobed for CASK to compare loading amounts (indicated as 15 µg and 30 µg). The bottom portion of the same membrane was probed for GFP. (C) Cells producing HERG870X were lysed and subjected to overnight treatment with EndoH and PNGase. Treated (+) and untreated (–) cell lysates were separated on SDS-PAGE and subjected to immunoblotting with an anti-Myc antibody (-Myc). The arrow indicates the position of the mature band (M). (D) Cells transfected with the cDNAs encoding HERGwt and HERG750-860 were lysed 1 day after transfection and treated with EndoH (+) or untreated (–). Equal amounts of solubilized proteins were subjected to immunoblotting with an anti-HA antibody. (E) Cells producing different mutant constructs were co-transfected with GFP, lysed and subjected to immunoblotting. (top) The membrane was probed with anti-HA and anti-CASK antibodies. (bottom) The same membrane was probed with an anti-GFP antibody. The arrow indicates the position of CASK signal at the expected molecular weight.

View larger version (48K): [in a new window]   Fig. 5. An intact CNBD is specifically involved in trafficking of HERG. (A) Immunoblot performed on HEK cells expressing cDNAs encoding HERGwt and HERG750-870. (B) HL-1 cardiac, PC12 neuronal and Cos7 kidney cells transfected with the indicated constructs and subjected to immunoblotting. (C) Immunocytochemical analyses of cells permeabilized with Triton X-100. Staining was performed with an anti-HA antibody followed by incubation with a secondary antibody conjugated with Oregon Green. Punctate staining of the HERGwt channel is absent from cells transfected with HERG750-870. (D) Cells expressing the HERG750-870 construct were treated with PK followed by immunoblotting. (top) The membrane was blotted with an anti-HA antibody, stripped and reprobed for CASK (bottom). Signal intensities before and after PK treatment were measured and quantified. (E) Immunoblot of cell extracts expressing HERG26-135 and HERG750-870 mutant channels. Membrane was probed with an anti-HERG antibody.

View larger version (28K): [in a new window]   Fig. 6. CNBD is essential for trafficking of ERG3. (A) Immunoblot analyses of cells transfected with ERG3wt before and after treatment with PNGase, EndoH or tunicamycin. The mature (M) and immature (I) immunoreactive species are shown by arrows. Tunicamycin treatment was performed overnight 1 day after transfection. (B) Pulse-chase analysis of ERG3wt at indicated chase times. Cells producing ERG3wt were labeled with radioactive cysteine and methionine, and chased at the indicated times. At the end of each chase interval, equal amounts of cell extracts were subjected to immunoprecipitation with an anti-HA antibody. The immunoprecipitated material was subjected to SDS-PAGE separation and autoradiography. Notice the gradual appearance of the mature band, which coincides with the disappearance of the immature species. (C) Immunoblot of cell extracts expressing ERG3 mutant variants. Cell lysates were subjected to EndoH (+) treatment and probed with an anti-HA antibody.

View larger version (37K): [in a new window]   Fig. 7. CNBD is required for ER exit but not tetramer assembly. (A) Cells producing HCN2wt were treated with PK (+) or left untreated (–) and subjected to immunoblotting with an anti-Myc antibody. PK concentrations are shown on top of each lane. (B) Cells producing HCN2 mutant variants were lysed and subjected to immunoblotting with an anti-Myc antibody. (C) Cells producing HERGwt and HERG750-870 were lysed and fractionated on linear non-denaturing sucrose gradients, and samples from the indicated fractions were subjected to immunoblot analysis. Pixel intensities of signals were measured and normalized to the maximum signal intensity. HERGwt and HERG750-870 are indicated by and , respectively. The position of markers is shown by arrows (alcohol dehydrogenase and thyroglobulin, 150 kDa and 669 kDa, respectively). (D) Sucrose-gradient analysis of HCN2 as described for HERG in C.

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