Peter M. Vassilev

Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells

Several proteins implicated in the pathogenesis of polycystic kidney disease (PKD) localize to cilia. Furthermore, cilia are malformed in mice with PKD with mutations in TgN737Rpw (encoding polaris). It is not known, however, whether ciliary dysfunction occurs or is relevant to cyst formation in PKD. Here, we show that polycystin-1 (PC1) and polycystin-2 (PC2), proteins respectively encoded by Pkd1 and Pkd2, mouse orthologs of genes mutated in human autosomal dominant PKD, co-distribute in the primary cilia of kidney epithelium. Cells isolated from transgenic mice that lack functional PC1 formed cilia but did not increase Ca2+ influx in response to physiological fluid flow. Blocking antibodies directed against PC2 similarly abolished the flow response in wild-type cells as did inhibitors of the ryanodine receptor, whereas inhibitors of G-proteins, phospholipase C and InsP3 receptors had no effect. These data suggest that PC1 and PC2 contribute to fluid-flow sensation by the primary cilium in renal epithelium and that they both function in the same mechanotransduction pathway. Loss or dysfunction of PC1 or PC2 may therefore lead to PKD owing to the inability of cells to sense mechanical cues that normally regulate tissue morphogenesis.

Molecular Cloning and Characterization of a Channel-like Transporter Mediating Intestinal Calcium Absorption

Calcium is a major component of the mineral phase of bone and serves as a key intracellular second messenger. Postnatally, all bodily calcium must be absorbed from the diet through the intestine. Here we report the properties of a calcium transport protein (CaT1) cloned from rat duodenum using an expression cloning strategy in Xenopus laevis oocytes, which likely plays a key role in the intestinal uptake of calcium. CaT1 shows homology (75% amino acid sequence identity) to the apical calcium channel ECaC recently cloned from vitamin D-responsive cells of rabbit kidney and is structurally related to the capsaicin receptor and the TRP family of ion channels. Based on Northern analysis of rat tissues, a 3-kilobase CaT1 transcript is present in rat duodenum, proximal jejunum, cecum, and colon, and a 6.5-kilobase transcript is present in brain, thymus, and adrenal gland. In situ hybridization revealed strong CaT1 mRNA expression in enterocytes of duodenum, proximal jejunum, and cecum. No signals were detected in kidney, heart, liver, lung, spleen, and skeletal muscle. When expressed inXenopus oocytes, CaT1 mediates saturable Ca2+uptake with a Michaelis constant of 0.44 mm. Transport of Ca2+ by CaT1 is electrogenic, voltage-dependent, and exhibits a charge/Ca2+uptake ratio close to 2:1, indicating that CaT1-mediated Ca2+ influx is not coupled to other ions. CaT1 activity is pH-sensitive, exhibiting significant inhibition by low pH. CaT1 is also permeant to Sr2+ and Ba2+ (but not Mg2+), although the currents evoked by Sr2+ and Ba2+ are much smaller than those evoked by Ca2+. The trivalent cations Gd3+ and La3+ and the divalent cations Cu2+, Pb2+, Cd2+, Co2+, and Ni2+ (each at 100 μm) do not evoke currents themselves, but inhibit CaT1-mediated Ca2+ transport. Fe3+, Fe2+, Mn2+, and Zn2+ have no significant effects at 100 μm on CaT1-mediated Ca2+ transport. CaT1 mRNA levels are not responsive to 1,25-dihydroxyvitamin D3 administration or to calcium deficiency. Our studies strongly suggest that CaT1 provides the principal mechanism for Ca2+ entry into enterocytes as part of the transcellular pathway of calcium absorption in the intestine.

Polycystin-L is a calcium-regulated cation channel permeable to calcium ions

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.

The Ca2+-sensing receptor: a target for polyamines

The Ca2+-sensing receptor (CaR) is activated at physiological levels of external Ca2+(Cao) but is expressed in a number of tissues that do not have well-established roles in the control of Cao, including several regions of the brain and the intestine. Polyamines are endogenous polyvalent cations that can act as agonists for the CaR, as shown by our current studies of human embryonic kidney (HEK-293) cells transfected with the human CaR. Cellular parameters altered by polyamines included cytosolic free Ca2+(Cai), inositol phosphate production, and the activity of a nonselective cation channel. Spermine stimulated Cai transients in CaR-transfected HEK cells, with a concentration producing a half-maximal response (EC50) of ∼500 μM in the presence of 0.5 mM Ca2+, whereas sustained increases in Cai had an EC50 of ∼200 μM. The order of potency was spermine > spermidine >> putrescine. Elevation of Cao shifted the EC50 for spermine sharply to the left, with substantial stimulation below 100 μM. Addition of subthreshold concentrations of spermine increased the sensitivity of CaR-expressing HEK cells to Cao. Parathyroid hormone secretion from bovine parathyroid cells was inhibited by 50% in the presence of 200 μM spermine, a response similar to that elicited by 2.0 mM Cao. These data suggest that polyamines could be effective agonists for the CaR, and several tissues, including the brain, may use the CaR as a target for the actions of spermine and other endogenous polycationic agonists.

Calcium ions as extracellular messengers.

The capacity to regulate their internal ionic composition is a fundamental property of free-living terrestrial organisms (Stewari and Broadus, 1987). This is accomplished by adjusting the movement of ions or water (or both) between cells and various extracellular compartments, including those communicating with the external environment. Of the complement of inorganic ions in such organisms, Ca2+ performs an especially large number of intracellular and extracellular functions. It is a well-known intracellular second messenger, controlling numerousvital processes(see minireview by Bootman and Berridge, 1995 [this issue of Ce//]). The cytosolic free Ca2+ concentration ([CaZ+li) undergoes rapid and often substantial (i.e., severalfold or more) fluctuations in response to extracellular first messengers binding to their cognate receptors on target cells. The extracellular ionized Ca2+ concentration ([CaZ+lO), in contrast, is maintained stably at 1 mM, at least lO,OOOfold higher than the resting level of [Ca2+li in most cells, providing a seemingly inexhaustible supply of Ca2+ for its diverse intracellular functions. Caz+ is also an essential component or cofactor in key extracellular structures or processes (i.e., bone, clotting factors, and adhesion molecules) whose biological functions could be compromised by large changes in [Ca2+10, with potentially disastrous consequences. It is not intuitively obvious, therefore, that Ca2+ serves as an important extracellular first messenger. The recent cloning of an extracellular CaZ+-sensing receptor (CaR), however, has firmly established that [Ca2+],, serves such an informational role (Brown et al., 1993). This minireview will briefly outline the signaling function of [Ca2+10, particularly the role of [CaQsensing mechanisms within various extracellular compartments in this process. The [Ca2+], Signal For extracellular Ca2+ to serve an informational role, it must undergo sufficiently large perturbations to be recognized by [Ca2+],-sensing mechanisms (or the responsiveness of the latter must change). In mammals, blood-ionized Ca*+ varies by only a few percent over the course of a day or even much of a lifetime. This remarkable stability of [CaZ+], results from a homeostatic system with two principal elements: [CaZ+],-sensing cells (e.g., parathyroid cells) and tissues that respond to either local or systemic signals from these [Ca2+],-sensing cells by altering their translocation of Ca2+ into or out of the extracellular fluid (i.e., kidney, intestine, and bone) (Stewart and Broadus, 1987). The near constancy of the blood-ionized CaZ+ concentration underscores the exquisite responsiveness of [Ca2+10Minireview

Native Polycystin 2 Functions as a Plasma Membrane Ca2+-Permeable Cation Channel in Renal Epithelia

ABSTRACT Mutations in polycystin 2 (PC2), a Ca2+-permeable cation channel, cause autosomal dominant polycystic kidney disease. Whether PC2 functions in the endoplasmic reticulum (ER) or in the plasma membrane has been controversial. Here we generated and characterized a polyclonal antibody against PC2, determined the subcellular localization of both endogenous and transfected PC2 by immunohistochemistry and biotinylation of cell surface proteins, and assessed PC2 channel properties with electrophysiology. Endogenous PC2 was found in the plasma membrane and the primary cilium of mouse inner medullar collecting duct (IMCD) cells and Madin-Darby canine kidney (MDCK) cells, whereas heterologously expressed PC2 showed a predominant ER localization. Patch-clamping of IMCD cells expressing endogenous or heterologous PC2 confirmed the presence of the channel on the plasma membrane. Treatment with chaperone-like factors facilitated the translocation of the PC2 channel to the plasma membrane from intracellular pools. The unitary conductances, channel kinetics, and other characteristics of both endogenously and heterologously expressed PC2 were similar to those described in our previous study in Xenopus laevis oocytes. These results show that PC2 functions as a plasma membrane channel in renal epithelia and suggest that PC2 contributes to Ca2+ entry and transport of other cations in defined nephron segments in vivo.

Sodium and Ionic Strength Sensing by the Calcium Receptor

The calcium-sensing receptor (CaR) is activated by small changes in extracellular calcium [Ca2+] o ) in the physiological range, allowing the parathyroid gland to regulate serum [Ca2+] o ; however, the CaR is also distributed in a number of other tissues where it may sense other endogenous agonists and modulators. CaR agonists are polycationic molecules, and charged residues in the extracellular domain of the CaR appear critical for receptor activation through electrostatic interactions, suggesting that ionic strength could modulate CaR activation by polycationic agonists. Changes in the concentration of external NaCl potently altered the activation of the CaR by external Ca2+ and spermine. Ionic strength had an inverse effect on the sensitivity of CaR to its agonists, with lowering of ionic strength rendering the receptor more sensitive to activation by [Ca2+] o and raising of ionic strength producing the converse effect. Effects of osmolality could not account for the modulation seen with changes in NaCl. Other salts, which differed in the cationic or anionic species, showed shifts in the activation of the CaR by [Ca2+] o similar to that elicited by NaCl. Parathyroid cells were potently modulated by ionic strength, with addition of 40 mm NaCl shifting the EC50 for [Ca2+] o inhibition of parathyroid hormone by at least 0.5 mm. Several CaR-expressing tissues, including regions of the brain such as the subfornical organ and hypothalamus, could potentially use the CaR as a sensor for ionic strength and NaCl. The Journal guidelines state that the summary should be no longer than 200 words.

Identification and characterization of the single channel function of human mucolipin-1 implicated in mucolipidosis type IV, a disorder affecting the lysosomal pathway

Mucolipin‐1 (MLN1) is a membrane protein with homology to the transient receptor potential channels and other non‐selective cation channels. It is encoded by the MCOLN1 gene, which is mutated in patients with mucolipidosis type IV (MLIV), an autosomal recessive disease that is characterized by severe abnormalities in neurological development as well as by ophthalmologic defects. At the cellular level, MLIV is associated with abnormal lysosomal sorting and trafficking. Here we identify the channel function of human MLN1 and characterize its properties. MLN1 represents a novel Ca2+‐permeable channel that is transiently modulated by changes in [Ca2+]. It is also permeable to Na+ and K+. Large unitary conductances were measured in the presence of these cations. With its Ca2+ permeability and modulation by [Ca2+], MLN1 could play a major role in Ca2+ transport regulating lysosomal exocytosis and potentially other phenomena related to the trafficking of late endosomes and lysosomes.

Amyloid-? proteins activate Ca2+-permeable channels through calcium-sensing receptors

The amyloid‐beta peptides (Aβ) are produced in excess in Alzheimers disease (AD) and may contribute to neuronal dysfunction and degeneration. This study provides strong evidence for a novel cellular target for the actions of Aβ, the phospholipase C‐coupled, extracellular Ca2+‐sensing receptor (CaR). We demonstrate that Aβs produce a CaR‐mediated activation of a Ca2+‐permeable, nonselective cation channel (NCC), probably via elevation in cytosolic Ca2+ (Cai), in cultured hippocampal pyramidal neurons from normal rats and from wild type mice but not those from mice with targeted disruption of the CaR gene (CaR −/−). Aβs also activate NCC in CaR‐transfected but not in nontransfected human embryonic kidney (HEK293) cells. Thus aggregates of Aβ deposited on hippocampal neurons in AD could inappropriately activate the CaR, stimulating Ca2+‐permeable channels and causing sustained elevation of Cai with resultant neuronal dysfunction.

G-protein-coupled, extracellular ca2+-sensing receptor : A versatile regulator of diverse cellular functions

Publisher Summary This chapter examines the importance of G-protein-coupled, extracellular Ca 2+ -sensing receptor as a versatile regulator of diverse cellular functions. In the extracellular space, calcium is a cofactor for clotting factors, adhesion molecules, and other proteins. It also regulates neuronal excitability and is an essential component of the mineral phase of the skeleton. Bone provides both a structural framework protecting crucial bodily structures and enabling locomotion and a large reservoir of mineral ions that can be mobilized in times of need. The maintenance of near constancy of Ca 2+ in tetrapods necessitates that specific cells of the mineral ion homeostatic system detect and respond in a homeostatically appropriate manner to the changes in plasma calcium concentration of the same order as its normal variability. Elevated levels of Ca 2+ also stimulate several aspects of osteoblast function in vitro that could promote increased bone formation in vivo and, therefore, reductions in Ca 2+ . The evidence for the presence of a G-protein-coupled CaR came from studies in bovine parathyroid cells examining the actions of Ca 2+ , the physiological agonist for the putative CaR, on intracellular second messengers. A variety of polyvalent cations mimic the actions of Ca 2+ on the osteoclast, but their pharmacological profile in this cell type differs distinctly from that in parathyroid and other CaR-expressing cells.