The multimeric structure of polycystin-2 (TRPP2): structural-functional correlates of homo- and hetero-multimers with TRPC1.

Polycystin-2 (PC2, TRPP2), the gene product of PKD2, whose mutations cause autosomal dominant polycystic kidney disease (ADPKD), belongs to the superfamily of TRP channels. PC2 is a non-selective cation channel, with multiple subconductance states. In this report, we explored structural and functional properties of PC2 and whether the conductance substates represent monomeric contributions to the channel complex. A kinetic analysis of spontaneous channel currents of PC2 showed that four intrinsic, non-stochastic subconductance states, which followed a staircase behavior, were both pH- and voltage-dependent. To confirm the oligomeric contributions to PC2 channel function, heteromeric PC2/TRPC1 channel complexes were also functionally assessed by single channel current analysis. Low pH inhibited the PC2 currents in PC2 homomeric complexes, but failed to affect PC2 currents in PC2/TRPC1 heteromeric complexes. Amiloride, in contrast, abolished PC2 currents in both the homomeric PC2 complexes and the heteromeric PC2/TRPC1 complexes, thus PC2/TRPC1 complexes have distinct functional properties from the homomeric complexes. The topological features of the homomeric PC2-, TRPC1- and heteromeric PC2/TRPC1 channel complexes, assessed by atomic force microscopy, were consistent with structural tetramers. TRPC1 homomeric channels had different average diameter and protruding height when compared with the PC2 homomers. The contribution of individual monomers to the PC2/TRPC1 hetero-complexes was easily distinguishable. The data support tetrameric models of both the PC2 and TRPC1 channels, where the overall conductance of a particular channel will depend on the contribution of the various functional monomers in the complex.

Cation channel activity of mucolipin-1: the effect of calcium.

Mucolipidosis type IV (MLIV) is a rare, neurogenetic disorder characterized by developmental abnormalities of the brain, and impaired neurological, ophthalmological, and gastric function. Considered a lysosomal disease, MLIV is characterized by the accumulation of large vacuoles in various cell types. Recent evidence indicates that MLIV is caused by mutations in MCOLN1, the gene that encodes mucolipin-1 (ML1), a 65-kDa protein showing sequence homology and topological similarities with polycystin-2 and other transient receptor potential (TRP) channels. In this report, our observations on the channel properties of ML1, and molecular pathophysiology of MLIV are reviewed and expanded. Our studies have shown that ML1 is a multiple sub-conductance, non-selective cation channel. MLIV-causing mutations result in functional differences in the channel protein. In particular, the V446L and DeltaF408 mutations retain channel function but have interesting functional differences with regards to pH dependence and Ca(2+) transport. While the wild-type protein is inhibited by Ca(2+) transport, mutant ML1 is not. Atomic force microscopy imaging of ML1 channels shows that changes in pH modify the aggregation and size of the ML1 channels, which has an impact on vesicular fusogenesis. The new evidence provides support for a novel role of ML1 cation channels in vesicular acidification and normal endosomal function.

Effect of hydro-osmotic pressure on polycystin-2 channel function in the human syncytiotrophoblast.

Polycystin-2 (PC2), one of the gene products whose mutations cause autosomal dominant polycystic kidney disease is a transient receptor potential (TRP)-type (TRPP2) Ca(2+)-permeable, non-selective cation channel. PC2 is localized in the plasma membrane, the primary cilium, and other cellular organelles of renal epithelial and other cells. Recent studies indicate that PC2 is involved in signal transduction events associated with the transient increase in cytosolic Ca(2+). Proof of evidence now hinges on involvement of the PC2 channel in the transduction of environmental signals. PC2 is abundantly expressed in the apical membrane of human syncytiotrophoblast (hST), a highly intricate epithelial tissue, which is essential for the maternal-fetal transfer of solutes, including ions. Physical forces such as hydrostatic (H) and osmotic (Pi) pressure play important roles in placenta homeostasis. In this study, we provide new information on PC2 channel regulation in the hST by these environmental factors, and propose a model as to how they may trigger the activation of PC2. Using apical hST vesicles reconstituted in a lipid bilayer system, we found that a change in either H or Pi modified PC2 channel activity. This stimulatory effect was no longer observed in hST vesicles pre-treated with the actin cytoskeleton disrupter cytochalasin D. As shown by immunofluorescence analysis PC2 co-localized with actin filaments in the vicinity of the plasma membrane. This co-localization was disrupted by cytochalasin D. Taken together, our findings indicate that physical forces exerted on cells regulate PC2 channel activity by a sensory mechanism involving the actin cytoskeleton.

Cytoskeletal regulation of calcium-permeable cation channels in the human syncytiotrophoblast: role of gelsolin.

The human syncytiotrophoblast (hST) is the most apical epithelial barrier that covers the villous tree of the human placenta. An intricate and highly organized network of cytoskeletal structures supports the hST. Recently, polycystin-2 (PC2), a TRP-type nonselective cation channel, was functionally observed in hST, where it may be an important player to Ca2+ transport. Little is known, however, about channel regulation in hST. In this report, the regulatory role of actin dynamics on PC2 channels reconstituted from hST apical membranes was explored. Acute addition of cytochalasin D (CD, 5 microg ml-1) to reconstituted hST apical membranes transiently increased K+ -permeable channel activity. The actin-binding proteins alpha-actinin and gelsolin, as well as PC2, were observed by Western blot and immunofluorescence analyses in hST vesicles. CD treatment of hST vesicles resulted in a re-distribution of actin filaments, in agreement with the effect of CD on K+ channel activity. In contrast, addition of exogenous monomeric actin, but not prepolymerized actin, induced a rapid inhibition of channel function in hST. This inhibition was obliterated by the presence of CD in the medium. The acute (<15 min) CD stimulation of K+ channel activity was mimicked by addition of the actin-severing protein gelsolin in the presence, but not in the absence, of micromolar Ca2+. Ca2+ transport through PC2 triggers a regulatory feedback mechanism, which is based on the severing and re-formation of filamentous actin near the channels. Cytoskeletal structures may thus be relevant to ion transport regulation in the human placenta.

Molecular pathophysiology of mucolipidosis type IV: pH dysregulation of the mucolipin-1 cation channel.

Mucolipidosis type IV (MLIV) is an autosomal recessive neurogenetic disorder characterized by developmental abnormalities of the brain and impaired neurological, ophthalmologic and gastric function. Large vacuoles accumulate in various types of cells in MLIV patients. However, the pathophysiology of the disease at the cellular level is still unknown. MLIV is caused by mutations in a recently described gene, MCOLN1, encoding mucolipin-1 (ML1), a 65 kDa protein whose function is also unknown. ML1 shows sequence homology and topological similarities with polycystin-2 and other transient receptor potential (Trp) channels. In this study, we assessed both, whether ML1 has ion channel properties, and whether disease-causing mutations in MCOLN1 have functional differences with the wild-type (WT) protein. ML1 channel function was assessed from endosomal vesicles of null (MCOLN1(-/-)) and ML1 over-expressing cells, and liposomes containing the in vitro translated protein. Evidence from both preparations indicated that WT ML1 is a multiple subconductance non-selective cation channel whose function is inhibited by a reduction of pH. The V446L and DeltaF408 MLIV causing mutations retain channel function but not the sharp inhibition by lowering pH. Atomic force imaging of ML1 channels indicated that changes in pH modified the aggregation of unitary channels. Mutant-ML1 did not change in size on reduction of pH. The data indicate that ML1 channel activity is regulated by a pH-dependent mechanism that is deficient in some MLIV causing mutations of the gene. The evidence also supports a novel role for cation channels in the acidification and normal endosomal function.

Polycystin-1 activates and stabilizes the polycystin-2 channel.

Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent genetic disorder largely caused by mutations in the PKD1 and PKD2 genes that encode the transmembrane proteins polycystin-1 and -2, respectively. Both proteins appear to be involved in the regulation of cell growth and maturation, but the precise mechanisms are not yet well defined. Polycystin-2 has recently been shown to function as a Ca(2+)-permeable, non-selective cation channel. Polycystin-2 interacts through its cytoplasmic carboxyl-terminal region with a coiled-coil motif in the cytoplasmic tail of polycystin-1 (P1CC). The functional consequences of this interaction on its channel activity, however, are unknown. In this report, we show that P1CC enhanced the channel activity of polycystin-2. R742X, a disease-causing polycystin-2 mutant lacking the polycystin-1 interacting region, fails to respond to P1CC. Also, P1CC containing a disease-causing mutation in its coiled-coil motif loses its stimulatory effect on wild-type polycystin-2 channel activity. The modulation of polycystin-2 channel activity by polycystin-1 may be important for the various biological processes mediated by this molecular complex.

Voltage dependence and pH regulation of human polycystin-2-mediated cation channel activity.

Polycystin-2, the product of the human PKD2 gene, whose mutations cause autosomal dominant polycystic kidney disease, is a large conductance, Ca(2+)-permeable non-selective cation channel. Polycystin-2 is functionally expressed in the apical membrane of the human syncytiotrophoblast, where it may play a role in the control of fetal electrolyte homeostasis. Little is known, however, about the mechanisms that regulate polycystin-2 channel function. In this study, the role of pH in the regulation of polycystin-2 was assessed by ion channel reconstitution of both apical membranes of human syncytiotrophoblast and the purified FLAG-tagged protein from in vitro transcribed/translated material. A kinetic analysis of single channel currents, including dwell time histograms, confirmed two open and two close states for spontaneous channel behavior and a strong voltage dependence of the open probability of the channel (P(o)). A reduction of cis pH (pH(cis)) decreased P(o) and shifted the voltage dependence of channel function but had no effect on the single channel conductance. An increase in pH(cis), in contrast, increased NP(o) (channel number times P(o)). Elimination of the H(+) chemical gradient did not reverse the low pH(cis) inhibition of polycystin-2. Similar findings confirmed the pH effect on the in vitro translated, FLAG-tagged purified polycystin-2. The data indicate the presence of an H(+) ion regulatory site in the channel protein, which is accessible from the cytoplasmic side of the protein. This protonation site controls polycystin-2 cation-selective channel activity.