Apical sorting of a voltage- and Ca ²⁺ -activated K ⁺ channel α-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation

Abstract

The voltage-and Ca 2؉ -activated K ؉ (KV,Ca) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissuedependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel KV,Ca ␣-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200-to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., KV,Ca ␤-subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells.human Slowpoke channel ͉ epithelial polarity

The Cytoplasmic Tail of Large Conductance, Voltage- and Ca2+-activated K+ (MaxiK) Channel Is Necessary for Its Cell Surface Expression

“…2 and 4. Bravo-Zehnder et al (45) also reported that myc-tagged MaxiK channel has characteristics similar to those of wild type. N terminus GFP tagging of hslo changes the channel's electrophysiological properties (46).…”

“…Using single channel patch recording, currents from MaxiK channels were recorded at apical or basolateral membranes of different cell types (23, 56 -63). Bravo-Zehnder et al (45) reported that when transfected into Madin-Darby canine kidney cells, hslo protein traffics to the apical membrane. Endogenous rabbit MaxiK channel localizes at the apical membranes of collapsed thick ascending limb of the loop of Henle, as shown using indirect immunofluorescence microscopy (12).…”

Sorting Competition with Membrane-permeable Peptides in Intact Epithelial Cells Revealed Discrimination of Transmembrane Proteins Not Only at the trans-Golgi Network but Also at Pre-Golgi Stages

“…Transfections with GtsO45 and G-EGFRc cDNAs were made using the LipofectAMINE Plus (Invitrogen) method according to the manufacturer's protocol, and permanent transformants were obtained after 2 weeks of selection with G418 (0.8 mg/ml) (Invitrogen) (49,50). Protein expression was induced by 10 mM sodium butyrate (49,50) at the restrictive temperature of 40°C for 20 h to accumulate the model proteins in the ER.…”

“…Domain-specific Biotinylation and Immunoprecipitation-Domainspecific biotinylation was made in MDCK cells cultured in Transwell chambers (49). The cells were washed twice in ice-cold PBS supplemented with 0.1 mM CaCl 2 and 1 mM MgCl 2 (PBS-CM), and then sulfo-NHS-biotin was added to a final concentration of 0.5 mg/ml for 60 min.…”

Slo1 Caveolin-binding Motif, a Mechanism of Caveolin-1-Slo1 Interaction Regulating Slo1 Surface Expression

“…5, G and H). The hSlo1 intracellular carboxyl-terminal end is known to play a role in Slo1 surface expression (33)(34)(35). The 1007 YNMLCFGIY 1015 sequence has several important features that may explain why its absence causes channel trapping inside the cell: (i) it has a di-hydrophobic motif (underlined), which may act as endoplasmic reticulum export signal (36), (ii) it has a targeting tyrosinebased motif YXX (bold; X, any amino acid and , hydrophobic residue), which is a general sorting sequence that directs proteins to various cellular compartments, including the plasma membrane (37), and (iii) it is located in a strategic position near another two sequences containing di-hydrophobic motifs, 996 YGKDFCKALK 1005 (in hSlo1, amino acids KD are replaced with DL, which add another dihydrophobic motif and a tyrosine-based motif) and 1047 DLIFCL 1052 , whose deletion is sufficient to prevent current development (38) and protein surface expression (35), respectively.…”