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First published online 17 August 2004
doi: 10.1242/jcs.01318

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Basolateral localisation of KCNQ1 potassium channels in MDCK cells: molecular identification of an N-terminal targeting motif

Thomas Jespersen^1,*, Hanne B. Rasmussen^1,*,, Morten Grunnet^1,3, Henrik S. Jensen¹, Kamilla Angelo¹, Delphine S. Dupuis¹, Lotte K. Vogel², Nanna K. Jorgensen¹, Dan A. Klaerke¹ and Søren-Peter Olesen^1,3

¹ Department of Medical Physiology and Copenhagen Heart Research Center, The Panum Institute, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark
² Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark
³ NeuroSearch A/S, Pederstrupvej 93, 2750 Ballerup, Denmark

View larger version (59K): [in a new window]   Fig. 1. Functional KCNQ1 channels are located in the basolateral membrane of MDCK cells. (A) Confocal images of MDCK cells transiently transfected with KCNQ1 cDNA. The KCNQ1 antibody stains at the basolateral plasma membrane (a1,a2). The basolateral membranes were selectively biotinylated (b1,b2) in order to visualise the polarisation of the cells and the localisation of the basolateral plasma membranes. (a1-c1) A single horizontal confocal scan through the cell layer close to the basal level; (a2-c2) vertical confocal scans of the same cells. (c) Overlays of the images in a,b. Ap, apical membrane, Ba, basal membrane. Bar, 8 µm. (B) Whole-cell electrophysiology on MDCK cells. Transfected cells were held at –80 mV, subjected to 2-second depolarising pulses ranging from –80 mV to +60 mV, followed by 1-second pulses at –30 mV. As expected, KCNQ1-expressing cells showed a slowly activating voltage-dependent current at potentials more positive than –40 mV. The obtained current could be blocked by 100 µM XE991, confirming that the current is carried by a KCNQ channel. The amplitude at the end of each 2-second pulse is illustrated in a current-voltage diagram (296 pA±52 pA at +60 mV, n=5).

View larger version (42K): [in a new window]   Fig. 2. KCNE proteins are not determinants for apical and/or basolateral targeting of the KCNQ1 channel. (A) MDCK cells transiently transfected with KCNQ1+KCNE1 (Q1+E1), KCNQ1+KCNE2 (Q1+E2), KCNQ1+KCNE3 (Q1+E3), KCNQ1+KCNE4 (Q1+E4) or KCNQ1+KCNE5 (Q1+E5). The cells were fixed and labeled for KCNQ1. The top panel shows horizontal confocal scans through the cell layer at the basal level, whereas the bottom panel shows vertical confocal scans of the same cells. Ap, apical membrane, Ba, basal membrane. Bar, 8 µm. (B) Verification of protein expression of KCNE1-5 and KCNQ1 was performed by electrophysiological measurements. Whole-cell patch-clamp analyses were done as described for KCNQ1 alone, except for KCNQ1/KCNE5, where the depolarising pulses were extended to +140 mV.

View larger version (25K): [in a new window]   Fig. 3. The N-terminal sequence of the KCNQ1 protein. Illustration of the truncation and deletion mutants constructed. An arrow indicates at which residue the sequence starts after the initiation methionine. Broken lines indicate potential sorting signals (LXL and YXX). The highlighted sequence indicates the fragment of 17 residues engrafted into p75NTR (see Fig. 8). The sKvLQT1 sequence is 95 residues shorter than wt-KCNQ1, indicated by 95, and the first 11 amino acids is non-homologous to wt-KCNQ1 (Neyroud et al., 1999).

View larger version (96K): [in a new window]   Fig. 4. Residues 20-40 of the KCNQ1 protein is important for steady-state localisation at the basolateral membrane. (A) Expression of wt-KCNQ1 and deletion mutants 20-KCNQ1 and 40-KCNQ1 in MDCK cells. The top panel shows a horizontal confocal scan through the cell layer at the basal level, whereas the bottom panel shows vertical confocal scans of the same cells. Bar, 8 µm. (B) The majority of sKvLQT1 staining in polarised MDCK cells is observed in intracellular compartments. Confocal scans of MDCK cells transfected with sKVLQT1 alone and cells co-transfected with sKvLQT1 and KCNE1. The fixed cells were labeled for sKvLQT1. Biotinylation of the apical membrane is applied (in green) to the vertical confocal scans. Ap, apical membrane, Ba, basal membrane. Bar, 8 µm.

View larger version (117K): [in a new window]   Fig. 5. The N-terminal LXL-motif is important for the steady-state plasma membrane localisation. (A) Confocal images of MDCK cells transfected with 2xL-A-KCNQ1 and double-labelled for KCNQ1 and markers of the endoplasmic reticulum (PDI) and the trans-Golgi network (TGN38). As illustrated, 2xL-A-KCNQ1 immunoreactivity is observed in close proximity to, but does not co-localize with that of the endoplasmic reticulum nor that of the Golgi apparatus. Ap, apical membrane, Ba, basal membrane. Bar, 4 µm. (B) MDCK cells transfected with 2xL-A-KCNQ1 were biotinylated at the basolateral membrane and chased at 37°C for 0 hours (a-c) or 1 hour (d-f). They were subsequently fixed (a-c) or treated with MesNa and then fixed (d-f), prior to permabilisation and double-labelling with the KCNQ1 antibody (a,d) and Alexa Fluor 488-streptavidin (b,e). After 1 hour of chase some of the KCNQ1-positive structures had been populated with internalised biotinylated plasma membrane proteins (yellow in f). Horizontal confocal scans through the cell layer at the basal level are shown in a1-f1, whereas a2-f2 show vertical confocal scans of the same cells. (c,f) Illustrated overlays of images a,b and images d,e, respectively.

View larger version (100K): [in a new window]   Fig. 6. Subcellular localisation of Y111A-KCNQ1 and Y51A-KCNQ1. MDCK cells transfected with Y111A-KCNQ1 (a-c) or Y51A-KCNQ1 (d-f) were biotinylated at the basolateral membrane (b) or the apical membrane (f2). The cells were fixed and labelled with KCNQ1 antibody (a,d,e,f1) and Alexa Fluor 488-streptavidin (b,f2). Horizontal confocal scans through the cell layer at the basal level are shown in a1-c1,d, whereas e shows a horizontal scan at the apical level of the same cells depicted in d. (a2-c2,f1-3) Vertical scans. (c,f3) Overlays of images a,b, and f1,f2, respectively. Y111A-KCNQ1 is located intracellularly, whereas Y51A-KCNQ1 displays a non-polarised distribution pattern in the plasma membrane. Ap, apical membrane, Ba, basal membrane. Bar, 8 µm.

View larger version (14K): [in a new window]   Fig. 7. Electrophysiological properties of 2xL-A-KCNQ1 and Y51A-KCNQ1. (A) Representative traces from MDCK cells expressing wt-KCNQ1, Y51A-KCNQ1, and 2xL-A-KCNQ1 channels. The traces were obtained after stepping from –80 mV to +60 mV for 2 seconds followed by 0.5 seconds at – 30 mV. (B) Mean current values of MDCK cells expressing wt-KCNQ1 or mutated KCNQ1. Peak current values, measured at +60 mV, were as follows: 296±52 (n=5) for wt-KCNQ1, 114±35 pA (n=8) for 2xL-A-KCNQ1, 265±70 pA (n=8) for Y51A-KCNQ1, 27±6 pA (n=5) for Y111A-KCNQ1, and 24±10 pA (n=4) for Y111C-KCNQ1.

View larger version (54K): [in a new window]   Fig. 8. Y51 is a dominant basolateral targeting signal. Expression of p75* and the chimeric constructs p75*-Q1(Y51) and p75*-Q1(Y51A) in MDCK cells. They were studied by confocal microscopy using a monoclonal antibody directed against p75. p75* is localised in the apical membrane of MDCK cells, whereas the chimeric construct p75*-Q1(Y51) is mainly located in the basolateral membrane. Mutation of Y51 into an alanine eliminates the ability of the KCNQ1 sequence to redirect p75* as p75*-Q1(Y51A) is located in the apical membrane. Both horizontal confocal scans through the cell layer and vertical confocal scans of the same cells are shown. For p75* and p75*-Q1(Y51A) horizontal scans through the cell layer at the apical level is illustrated, whereas a horizontal scan at the basal level is included for p75*-Q1(Y51). Ap, apical membrane, Ba, basal membrane. Bar, 8 µm.

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