A Transmembrane Segment Determines the Steady-State Localization of an Ion-Transporting Adenosine Triphosphatase

100

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

mentioning

confidence: 84%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Bravo‐Zehnder

Orio

Norambuena

et al. 2000

100

“…A number of studies have identified dominant sorting signals in the nonglycosylated catalytic ␣-subunit in Na ϩ ,K ϩ -ATPase and H ϩ ,K ϩ -ATPase (33,34) that are responsible for the basolateral or apical localization, respectively, of the heterodimeric pump. The basolateral localization of Na ϩ ,K ϩ -ATPase is additionally stabilized by interactions of the ␣-subunit with ankyrin at the lateral membrane (35) and by interactions between neighboring ␤-subunits (36).…”

Section: Discussionmentioning

confidence: 99%

Mechanisms regulating tissue-specific polarity of monocarboxylate transporters and their chaperone CD147 in kidney and retinal epithelia

Deora

Philp²,

Hu³

et al. 2005

126

121

Proton-coupled monocarboxylate transporters (MCT) MCT1, MCT3, and MCT4 form heterodimeric complexes with the cell surface glycoprotein CD147 and exhibit tissue-specific polarized distributions that are essential for maintaining lactate and pH homeostasis. In the parenchymal epithelia of kidney, thyroid, and liver, MCT͞CD147 heterocomplexes are localized in the basolateral membrane where they transport lactate out of or into the cell depending on metabolic conditions. A unique distribution of lactate transporters is found in the retinal pigment epithelium (RPE), which regulates lactate levels of the outer retina. In RPE, MCT1͞ CD147 is polarized to the apical membrane and MCT3͞CD147 to the basolateral membrane. The mechanisms responsible for tissuespecific polarized distribution of MCTs are unknown. Here, we demonstrate that CD147 carries sorting information for polarized targeting of the MCT1͞CD147 hetero-complexes in kidney and RPE cells. In contrast, MCT3 and MCT4 harbor dominant sorting information that cotargets CD147 to the basolateral membrane in both epithelia. RNA interference experiments show that MCT1 promotes CD147 maturation. Our results open a unique paradigm to study the molecular basis of tissue-specific polarity.apical ͉ basolateral ͉ polarized epithelium

“…PDZ domains are modules for protein-protein interaction and are involved in sorting and͞or the stability of asymmetrically distributed proteins (30,31). The findings that NaPi-Cap1 and NHERF1 are expressed in the renal proximal BBM and interact with the C-terminal tail of NaPi IIa in a PDZ-motif-dependent manner, together with the observation that this terminal PDZmotif contains information for apical expression, prompted us to analyze whether such interactions are involved in apical expression of NaPi IIa.…”

mentioning

confidence: 99%

PDZ-domain interactions and apical expression of type IIa Na/P _i cotransporters

Hernando

Déliot

Gisler

et al. 2002

165

147

Type IIa Na͞Pi cotransporters are expressed in renal proximal brush border and are the major determinants of inorganic phosphate (Pi) reabsorption. Their carboxyl-terminal tail contains information for apical expression, and interacts by means of its three terminal amino acids with several PSD95͞DglA͞ZO-1-like domain (PDZ)-containing proteins. Two of these proteins, NaPi-Cap1 and Na͞H exchanger-regulatory factor 1 (NHERF1), colocalize with the cotransporter in the proximal brush border. We used opossum kidney cells to test the hypothesis of a potential role of PDZ-interactions on the apical expression of the cotransporter. We found that opossum kidney cells contain NaPi-Cap1 and NHERF1 mRNAs. For NHERF1, an apical location of the protein could be documented; this location probably reflects interaction with the cytoskeleton by means of the MERM-binding domain. Overexpression of PDZ domains involved in interaction with the cotransporter (PDZ-1͞ NHERF1 and PDZ-3͞NaPi-Cap1) had a dominant-negative effect, disturbing the apical expression of the cotransporter without affecting the actin cytoskeleton or the basolateral expression of Na͞K-ATPase. These data suggest an involvement of PDZ-interactions on the apical expression of type IIa cotransporters.opossum kidney cells ͉ Na͞H exchanger-regulatory factor 1 ͉ proximal tubules P roximal tubular reabsorption of inorganic phosphate (P i ) plays a key role in P i metabolism (1, 2). Up to 80% of the renal reabsorption of P i is mediated by the brush border membrane (BBM)-associated type IIa Na͞P i -cotransporters (NaPi IIa; refs. 3 and 4; for review, see ref.2). According to their key physiological role, these cotransporters are up-regulated by factors that stimulate renal reabsorption of P i (ref. 5; for review see ref.2), whereas they are down-regulated by phosphaturic factors (refs. 6 and 7; for review, see ref. 2). Their expression is also affected in pathological states associated with P i wasting, such as X-linked hypophosphatemic rickets: a primary defect on the PHEX gene leads by a yet-unknown mechanism to a reduced expression of NaPi IIa (8, 9). Many of the proximal tubular characteristics in terms of P i handling are retained in a cell line derived from opossum kidney (OK) cells. These OK cells contain an endogenous NaPi IIa cotransporter (NaPi4) apically located and regulated by the same hormones and factors as NaPi IIa cotransporters in proximal tubules (6,10,11).NaPi IIa cotransporters are predicted to contain eight transmembrane domains with intracellular N-and C-terminal tails (12). The C-terminal tail contains two signals involved in apical expression: a terminal PSD95͞DglA͞ZO-1-like domain (PDZ)-binding motif (TRL) and an internal determinant (13). The C-terminal tail interacts, by means of TRL residues, with several PDZ-containing proteins, among them NaPi-Cap1 and Na͞H exchanger-regulatory factor 1 (NHERF1; ref. 14). Similar to NaPi IIa, both proteins are located on the BBM of proximal tubules (14, 15). NaPi-Cap1 is a protein of about 500 residues that co...