Polycystic kidney diseases are genetic disorders in which the renal parenchyma is progressively replaced by fluid-filled cysts. Two members of the polycystin family (polycystin-1 and -2) are mutated in autosomal dominant polycystic kidney disease (ADPKD), and polycystin-L is deleted in mice with renal and retinal defects. Polycystins are membrane proteins that share significant sequence homology, especially polycystin-2 and -L (50% identity and 71% similarity). The functions of the polycystins remain unknown. Here we show that polycystin-L is a calcium-modulated nonselective cation channel that is permeable to sodium, potassium and calcium ions. Patch-clamp experiments revealed single-channel activity with a unitary conductance of 137 pS. Channel activity was substantially increased when either the extracellular or intracellular calcium-ion concentration was raised, indicating that polycystin-L may act as a transducer of calcium-mediated signalling in vivo. Its large single-channel conductance and regulation by calcium ions distinguish it from other structurally related cation channels.

Mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene have been shown to cause autosomal recessive polycystic kidney disease (ARPKD), but the cellular functions of the gene product (PKHD1) remain uncharacterized. To illuminate its properties, the spatial and temporal expression patterns of PKHD1 were determined in mouse, rat, and human tissues by using polyclonal Abs and mAbs recognizing various specific regions of the gene product. During embryogenesis, PKHD1 is widely expressed in epithelial derivatives, including neural tubules, gut, pulmonary bronchi, and hepatic cells. In the kidneys of the pck rats, the rat model of which is genetically homologous to human ARPKD, the level of PKHD1 was significantly reduced but not completely absent. In cultured renal cells, the PKHD1 gene product colocalized with polycystin-2, the gene product of autosomal dominant polycystic disease type 2, at the basal bodies of primary cilia. Immunoreactive PKHD1 localized predominantly at the apical domain of polarized epithelial cells, suggesting it may be involved in the tubulogenesis and͞or maintenance of duct-lumen architecture. Reduced PKHD1 levels in pck rat kidneys and its colocalization with polycystins may underlie the pathogenic basis for cystogenesis in polycystic kidney diseases.

Autosomal recessive polycystic kidney disease is caused by mutations in PKHD1, which encodes the membrane-associated receptor-like protein fibrocystin/polyductin (FPC). FPC associates with the primary cilia of epithelial cells and co-localizes with the Pkd2 gene product polycystin-2 (PC2), suggesting that these two proteins may function in a common molecular pathway. For investigation of this, a mouse model with a gene-targeted mutation in Pkhd1 that recapitulates phenotypic characteristics of human autosomal recessive polycystic kidney disease was produced. The absence of FPC is associated with aberrant ciliogenesis in the kidneys of Pkhd1-deficient mice. It was found that the COOH-terminus of FPC and the NH2-terminus of PC2 interact and that lack of FPC reduced PC2 expression but not vice versa, suggesting that PC2 may function immediately downstream of FPC in vivo. PC2-channel activities were dysregulated in cultured renal epithelial cells derived from Pkhd1 mutant mice, further supporting that both cystoproteins function in a common pathway. In addition, mice with mutations in both Pkhd1 and Pkd2 had a more severe renal cystic phenotype than mice with single mutations, suggesting that FPC acts as a genetic modifier for disease severity in autosomal dominant polycystic kidney disease that results from Pkd2 mutations. It is concluded that a functional and molecular interaction exists between FPC and PC2 in vivo.

Proton-coupled peptide transporters mediate the absorption of a large variety of di-and tripeptides as well as peptide-like pharmacologically active compounds. We report a kinetic analysis of the rat kidney highaffinity peptide transporter PepT2 expressed in Xenopus oocytes. By use of simultaneous radioactive uptake and current measurements under voltage-clamp condition, the charge to substrate uptake ratio was found to be close to 2 for both D-Phe-L-Ala and D-Phe-L-Glu, indicating that the H ؉ :substrate stoichiometry is 2:1 and 3:1 for neutral and anionic dipeptides, respectively. The higher stoichiometry for anionic peptides suggests that they are transported in the protonated form. For D-Phe-L-Lys, the charge:uptake ratio averaged 2.4 from pooled experiments, suggesting that Phe-Lys crosses the membrane via PepT2 either in its deprotonated (neutral) or its positively charged form, averaging a H ؉ :Phe-Lys stoichiometry of 1.4:1. These findings led to the overall conclusion that PepT2 couples transport of one peptide molecule to two H ؉ . This is in contrast to the low-affinity transporter PepT1 that couples transport of one peptide to one H ؉ . Quinapril inhibited PepT2-mediated currents in presence or in absence of external substrates. Oocytes expressing PepT2 exhibited quinapril-sensitive outward currents. In the absence of external substrate, a quinapril-sensitive proton inward current (proton leak) was also observed which, together with the observed pH-dependent PepT2-specific presteady-state currents (I pss ), indicates that at least one H ؉ binds to the transporter prior to substrate. PepT2 exhibited I pss in response to hyperpolarization at pH 6.5-8.0. However, contrary to previous observations on various transporters, 1) no significant currents were observed corresponding to voltage jumps returning from hyperpolarization, and 2) at reduced extracellular pH, no significant I pss were observed in either direction. Together with observed lower substrate affinities and decreased PepT2-mediated currents at hyperpolarized V m , our data are consistent with the concept that hyperpolarization exerts inactivation effects on the transporter which are enhanced by low pH. Our studies revealed distinct properties of PepT2, compared with PepT1 and other ion-coupled transporters.In kidney and intestine, enzymatic degradation of proteins and peptides produces oligopeptides that are absorbed across the brush-border membrane of epithelial cells, followed by breakdown into free amino acids (1-3). The absorption is carried out by proton-driven cotransport systems, as demonstrated by studies using brush-border membrane vesicles, epithelial cells in culture, intact epithelial tubules, and recombined peptide transporters (4 -10). A large variety of diand tripeptides, as well as pharmacologically active peptidelike compounds such as ␤-lactam antibiotics and angiotensinconverting enzyme inhibitors, are transported (11-13). Peptide transporters have been cloned since 1994 (3, 14 -19) and were shown to accept many oligopeptide...

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Transient receptor potential (TRP) channels are regulated by diverse stimuli comprising thermal, chemical, and mechanical modalities. They are also commonly regulated by phosphatidylinositol-4,5-bisphosphate (PIP2), with underlying mechanisms largely unknown. We here revealed an intramolecular interaction of the TRPP3 N and C termini (N-C) that is functionally essential. The interaction was mediated by aromatic Trp81 in pre-S1 domain and cationic Lys568 in TRP-like domain. Structure-function analyses revealed similar N-C interaction in TRPP2 as well as TRPM8/-V1/-C4 via highly conserved tryptophan and lysine/arginine residues. PIP2 bound to cationic residues in TRPP3, including K568, thereby disrupting the N-C interaction and negatively regulating TRPP3. PIP2 had similar negative effects on TRPP2. Interestingly, we found that PIP2 facilitates the N-C interaction in TRPM8/-V1, resulting in channel potentiation. The intramolecular N-C interaction might represent a shared mechanism underlying the gating and PIP2 regulation of TRP channels.

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