Polycystin-2 (PC-2) is a non-selective cation channel that, when mutated, results in autosomal dominant polycystic kidney disease. In an effort to understand the regulation of this channel, we investigated the role of protein phosphorylation in PC-2 function. We demonstrated the direct incorporation of phosphate into PC-2 in cells and tissues and found that this constitutive phosphorylation occurs at Ser 812 , a putative casein kinase II (CK2) substrate domain. Ser 812 can be phosphorylated by CK2 in vitro and substitution S812A results in failure to incorporate phosphate in cultured epithelial cells. Non-phosphorylated forms of PC-2 traffic normally in the endoplasmic reticulum and cilial compartments and retain homo-and hetero-multimerization interactions with PC-2 and polycystin-1, respectively. Single-channel studies of PC-2, S812A, and a substitution mutant, T721A, not related to phosphorylation show that PC-2 and S812A function as divalent cation channels with similar current amplitudes across a range of holding potentials; the T721A channel is not functional. Channel open probabilities for PC-2 and S812A show a bell-shaped dependence on cytoplasmic Ca 2؉ but there is a shift in this Ca 2؉ dependence such that S812A is 10-fold less sensitive to Ca 2؉ activation/inactivation than the wild type PC-2 channel. In vivo analysis of PC-2-dependent enhanced intracellular Ca 2؉ transients found that S812A resulted in enhanced transient duration and relative amplitude intermediate between control cells and those overexpressing wild type PC-2. Phosphorylation at Ser 812 modulates PC-2 channel activity and factors regulating this phosphorylation are likely to play a role in the pathogenesis of polycystic kidney disease.

No abstract

Mutations in polycystin-2 (PC2) cause autosomal dominant polycystic kidney disease. A function for PC2 in the heart has not been described. Here, we show that PC2 coimmunoprecipitates with the cardiac ryanodine receptor (RyR2) from mouse heart. Biochemical assays showed that the N terminus of PC2 binds the RyR2, whereas the C terminus only binds to RyR2 in its open state. Lipid bilayer electrophysiological experiments indicated that the C terminus of PC2 functionally inhibited RyR2 channel activity in the presence of calcium (Ca 2؉ ). Pkd2 ؊/؊ cardiomyocytes had a higher frequency of spontaneous Ca 2؉ oscillations, reduced Ca 2؉ release from the sarcoplasmic reticulum stores, and reduced Ca 2؉ content compared with Pkd2 ؉/؉ cardiomyocytes. In the presence of caffeine, Pkd2 ؊/؊ cardiomyocytes exhibited decreased peak fluorescence, a slower rate of rise, and a longer duration of Ca 2؉ transients compared with Pkd2 ؉/؉ . These data suggest that PC2 is important for regulation of RyR2 function and that loss of this regulation of RyR2, as occurs when PC2 is mutated, results in altered Ca 2؉ signaling in the heart. intracellular calcium channel ͉ polycystic kidney disease ͉ lipid bilayer ͉ calcium imaging ͉ cardiac cells

Polycystin-2 (PC2), the gene product of one of two genes mutated in dominant polycystic kidney disease, is a member of the transient receptor potential cation channel family and can function as intracellular calcium (Ca 2؉ ) release channel. We performed a yeast two-hybrid screen by using the NH 2 terminus of PC2 and identified syntaxin-5 (Stx5) as a putative interacting partner. Coimmunoprecipitation studies in cell lines and kidney tissues confirmed interaction of PC2 with Stx5 in vivo. In vitro binding assays showed that the interaction between Stx5 and PC2 is direct and defined the respective interaction domains as the t-SNARE region of Stx5 and amino acids 5 to 72 of PC2. Single channel studies showed that interaction with Stx5 specifically reduces PC2 channel activity. Epithelial cells overexpressing mutant PC2 that does not bind Stx5 had increased baseline cytosolic Ca 2؉ levels, decreased endoplasmic reticulum (ER) Ca 2؉ stores, and reduced Ca 2؉ release from ER stores in response to vasopressin stimulation. Cells lacking PC2 altogether had reduced cytosolic Ca 2؉ levels. Our data suggest that PC2 in the ER plays a role in cellular Ca 2؉ homeostasis and that Stx5 functions to inactivate PC2 and prevent leaking of Ca 2؉ from ER stores. Modulation of the PC2/Stx5 interaction may be a useful target for impacting dysregulated intracellular Ca 2؉ signaling associated with polycystic kidney disease.Ca 2ϩ channel ͉ polycystic kidney disease ͉ t-SNARE ͉ TRP channel A utosomal dominant polycystic kidney disease (ADPKD) is characterized by the growth of cysts, occurring over decades, in previously normal appearing kidney tubules (1). A functional hallmark of ADPKD is the loss of a calcium (Ca 2ϩ ) signal that serves to inhibit dysregulated kidney tubule cell proliferation, polarization, and secretory function. Either of two causative genes, PKD1 or PKD2, can initiate cyst formation after homozygous loss-of-function mutations, typically resulting from a combination of germline mutation on one allele followed by somatic second step mutation occurring at the level of individual cells. The respective protein products, polycystin-1 (PC1) and polycystin-2 (PC2), form a receptor-channel complex in the membrane of the apical primary cilia in renal tubular cells (2, 3), as well as in bile and pancreatic duct cells. PC2 (TRPP2), a member of the transient receptor potential (TRP) cation channel family, is abundantly expressed in the endoplasmic reticulum (ER) membrane (4) and has been shown to function as a Ca 2ϩ release channel from intracellular stores (5, 6). Indirect evidence has led to the proposal that PC2 channel activity is required for the rise in cellular Ca 2ϩ in ciliated monolayers of cultured epithelial cells under conditions of laminar shear stress because of fluid flow (7,8). This response to flow requires Ca 2ϩ from both extracellular and ER stores (9).Although variations in the reported channel properties of PC2 exist, it is generally accepted that PC2 is a high conductance cation channel (40-177 pS) whose a...

Polycystin-2 (PC2), a member of the transient receptor potential family of ion channels (TRPP2), forms a calcium-permeable cation channel. Mutations in PC2 lead to polycystic kidney disease. From the primary sequence and by analogy with other channels in this family, PC2 is modeled to have six transmembrane domains. However, most of the structural features of PC2, such as how large the channel is and how many subunits make up the pore of the channel, are unknown. In this study, we estimated the pore size of PC2 from the permeation properties of the channel. Organic cations of increasing size were used as current carriers through the PC2 channel after PC2 was incorporated into lipid bilayers. We found that dimethylamine, triethylamine, tetraethylammonium, tetrabutylammonium, tetrapropylammonium, and tetrapentylammonium were permeable through the PC2 channel. The slope conductance of the PC2 channel decreased as the ionic diameter of the organic cation increased. For each organic cation tested, the currents were inhibited by gadolinium and anti-PC2 antibody. Using the dimensions of the largest permeant cation, the minimum pore diameter of the PC2 channel was estimated to be at least 11 Å. The large pore size suggests that the primary state of this channel found in vivo is closed to avoid rundown of cation gradients across the plasma membrane and excessive calcium leak from endoplasmic reticulum stores.

No abstract

The homotetrameric structure of the ryanodine-sensitive intracellular calcium (Ca 2؉ ) release channel (ryanodine receptor (RyR)) suggests that the four RyR subunits either combine to form a single pore or that each RyR subunit is an independently conducting pathway. Previously we showed that methanethiosulfonate ethylammonium (MTSEA ؉ ) covalently modifies the RyR to reduce current amplitudes in a time-dependent and stepwise manner. To ascertain the number of functionally conducting pores in the RyR, two approaches were combined: modification of the receptor by MTSEA ؉ and the use of different sized current carriers. Previous reports (Tinker, A., and Williams, A. J. (1993) J. Gen. Physiol. 102, 1107-1129) have shown that the organic cations methylamine, dimethylamine, ethylamine, and trimethylamine are permeant through the RyR but with reduced current amplitude depending upon the diameter of the respective amine. Experiments using the thiol reagent MTSEA ؉ to modify the channel protein showed that the current amplitudes decrease in steps leading to complete block of the channel when cesium (Cs ؉ ) is the current carrier. MTSEA ؉ modification decreased the number of channel substates as the diameter of the current carrier increased. Comparison of the degree of inhibition of MTSEA ؉ -modified currents allows for differentiation between the two models for channel architecture. These results demonstrate that the conduction pathway for the RyR is comprised of a single central pore.

We have used limited trypsin digestion and reactivity with PEG-maleimides (MPEG) to study Ca 2؉ -induced conformational changes of IP 3 Rs in their native membrane environment. We found that Ca 2؉ decreased the formation of the 95-kDa C-terminal tryptic fragment when detected by an Ab directed at a C-terminal epitope (CT-1) but not with an Ab recognizing a protected intraluminal epitope. This suggests that Ca 2؉ induces a conformational change in the IP 3 R that allows trypsin to cleave the C-terminal epitope. Half-maximal effects of Ca 2؉ were observed at ϳ0.5 M and was sensitive to inhibition by IP 3 . Ca 2؉ also stimulated the reaction of MPEG-5 with an endogenous thiol in the 95-kDa fragment. This effect was eliminated when six closely spaced cysteine residues proximal to the transmembrane domains were mutated (C2000S, C2008S, C2010S, C2043S, C2047S, and C2053S) or when the N-terminal suppressor domain (amino acids 1-225) was deleted. A cysteine substitution mutant introduced at the C-terminal residue (A2749C) was freely accessible to MPEG-5 or MPEG-20 in the absence of Ca 2؉ . However, cysteine substitution mutants in the interior of the tail were poorly reactive with MPEG-5, although reactivity was enhanced by Ca 2؉ . We conclude the following: a) that large conformational changes induced by Ca 2؉ can be detected in IP 3 Rs in situ; b) these changes may be driven by Ca 2؉ binding to the N-terminal suppressor domain and expose a group of closely spaced endogenous thiols in the channel domain; and c) that the C-terminal cytosol-exposed tail of the IP 3 R may be relatively inaccessible to regulatory proteins unless Ca 2؉ is present.Inositol 1,4,5-trisphosphate receptors (IP 3 Rs) 3 are large tetrameric intracellular Ca 2ϩ -release channels that mediate the release of Ca 2ϩ from endoplasmic reticulum stores into the cytosol in response to IP 3 (1-5). Three different isoforms exist, which can form homo-and heteroligomers. All three isoforms consist of an IP 3 binding domain in the N-terminal region, a channel domain containing six transmembrane segments in the C-terminal region and an intervening regulatory domain. Apart from IP 3 , the principal regulator of the channel is Ca 2ϩ , which exerts a biphasic effect on channel function with activation at low concentrations and inhibition at high concentrations. The structural organization of this complex protein and the mechanism by which binding of IP 3 and Ca 2ϩ are linked to channel gating are active areas of investigation.An important feature of the mechanism of the channel involves conformational changes in the protein. In particular, the gating mechanism is thought to involve conformational changes initiated by IP 3 binding that are propagated to various regions of the channel domain (6, 7). The IP 3 binding domain of the receptor is organized into a "core" segment that binds IP 3 with high affinity (amino acids 226 -604) and a "suppressor domain" that inhibits IP 3 binding to the core (amino acids 1-225). Crystal structures for the suppressor domain and the ...