Nephrotic syndrome, a malfunction of the kidney glomerular filter, leads to proteinuria, edema and, in steroid-resistant nephrotic syndrome, end-stage kidney disease. Using positional cloning, we identified mutations in the phospholipase C epsilon gene (PLCE1) as causing early-onset nephrotic syndrome with end-stage kidney disease. Kidney histology of affected individuals showed diffuse mesangial sclerosis (DMS). Using immunofluorescence, we found PLCepsilon1 expression in developing and mature glomerular podocytes and showed that DMS represents an arrest of normal glomerular development. We identified IQ motif-containing GTPase-activating protein 1 as a new interaction partner of PLCepsilon1. Two siblings with a missense mutation in an exon encoding the PLCepsilon1 catalytic domain showed histology characteristic of focal segmental glomerulosclerosis. Notably, two other affected individuals responded to therapy, making this the first report of a molecular cause of nephrotic syndrome that may resolve after therapy. These findings, together with the zebrafish model of human nephrotic syndrome generated by plce1 knockdown, open new inroads into pathophysiology and treatment mechanisms of nephrotic syndrome.

SUMMARY In the mammalian central nervous system, slow synaptic excitation involves the activation of metabotropic glutamate receptors (mGluRs). It has been proposed that C1-type transient receptor potential (TRPC1) channels underlie this synaptic excitation, but our analysis of TRPC1-deficient mice does not support this hypothesis. Here, we show unambiguously that it is TRPC3 that is needed for mGluR-dependent synaptic signaling in mouse cerebellar Purkinje cells. TRPC3 is the most abundantly expressed TRPC subunit in Purkinje cells. In mutant mice lacking TRPC3, both slow synaptic potentials and mGluR-mediated inward currents are completely absent, while the synaptically mediated Ca2+ release signals from intracellular stores are unchanged. Importantly, TRPC3 knockout mice exhibit an impaired walking behavior. Taken together, our results establish TRPC3 as a new type of postsynaptic channel that mediates mGluR-dependent synaptic transmission in cerebellar Purkinje cells and is crucial for motor coordination.

We demonstrate that phenylalanine ammonia‐lyase (PAL) in parsley (Petroselinum crispum) is encoded by a small family of at least four genes. The levels of mRNA from three identified PAL genes increase considerably upon treatment of cultured parsley cells with UV light or fungal elicitor and upon wounding of parsley leaves or roots. In cultured cells these changes were shown to involve transcriptional activation. We present the first primary structure of a plant PAL gene (parsley PAL‐1) and the deduced amino acid sequence of the enzyme. Inducible in vivo footprints in the PAL‐1 promoter define two nucleotide sequences, within the motifs CTCCAACAAACCCCTTC and ATTCTCACCTACCA, involved in the responses to both UV irradiation and elicitor application. These motifs are conserved at similar positions in several elicitor or light‐responsive genes from different species. In two cases they are found within short regions known to confer elicitor or UV‐light inducibility. The conserved motifs in the parsley 4‐coumarate:CoA ligase gene, which is coordinately regulated with PAL, also display UV‐light inducible in vivo footprints. Taken together, our findings suggest a general role of these putative cis‐acting elements in the responses of plants to such stresses.

Regional alveolar hypoxia causes local vasoconstriction in the lung, shifting blood flow from hypoxic to normoxic areas, thereby maintaining gas exchange. This mechanism is known as hypoxic pulmonary vasoconstriction (HPV). Disturbances in HPV can cause life-threatening hypoxemia whereas chronic hypoxia triggers lung vascular remodeling and pulmonary hypertension. The signaling cascade of this vitally important mechanism is still unresolved. Using transient receptor potential channel 6 (TRPC6)-deficient mice, we show that this channel is a key regulator of acute HPV as this regulatory mechanism was absent in TRPC6 ؊/؊ mice whereas the pulmonary vasoconstrictor response to the thromboxane mimetic U46619 was unchanged. Accordingly, induction of regional hypoventilation resulted in severe arterial hypoxemia in TRPC6 ؊/؊ but not in WT mice. This effect was mirrored by a lack of hypoxiainduced cation influx and currents in smooth-muscle cells from precapillary pulmonary arteries (PASMC) of TRPC6 ؊/؊ mice. In both WT and TRPC6 ؊/؊ PASMC hypoxia caused diacylglycerol (DAG) accumulation. DAG seems to exert its action via TRPC6, as DAG kinase inhibition provoked a cation influx only in WT but not in TRPC6 ؊/؊ PASMC. Notably, chronic hypoxia-induced pulmonary hypertension was independent of TRPC6 activity. We conclude that TRPC6 plays a unique and indispensable role in acute hypoxic pulmonary vasoconstriction. Manipulation of TRPC6 function may thus offer a therapeutic strategy for the control of pulmonary hemodynamics and gas exchange.hypoxia-induced diacylglycerol accumulation ͉ precapillary pulmonary arterial smooth-muscle cells ͉ pulmonary hypertension ͉ transient receptor potential channel 6-deficient mouse model ͉ arterial hypoxemia A cute regional hypoxic pulmonary vasoconstriction (HPV) is necessary to maintain optimized gas exchange by directing blood flow from poorly ventilated to well ventilated areas of the lung. Under conditions of generalized hypoxia, however, total pulmonary vascular resistance rises with subsequent increase of right heart load (1-3). Chronic hypoxia, as occurring in ventilatory disorders induces chronic pulmonary hypertension, pulmonary vascular remodeling, and cor pulmonale (4). The underlying oxygen sensing and signal transduction mechanisms of the acute and chronic vascular responses are largely unknown. A rise of intracellular calcium ([Ca 2ϩ ] i ) in pulmonary artery smooth-muscle cells (SMCs) has been suggested to be the key event in these processes (5-8). However, the question how [Ca 2ϩ ] i is regulated has not yet been resolved. Among others, transient receptor potential (TRP) channels are regulators of [Ca 2ϩ ] i . The TRP protein superfamily consists of a diverse group of nonselective cation channels involved in many basic cellular processes (9). Whereas members of the TRPV and TRPM subfamilies have emerged as versatile cellular sensors, the functional importance of the seven members (TRPC1 to -7) of the TRPC (transient receptor potential cation channel subfamily C) subfamily...

Homologues ofS timulation of cells that elevates inositol 1,4,5-trisphosphate (IP3) causes the release of Ca 2ϩ from internal stores and its entry from the external milieu (for reviews see refs. 1-3). The release from internal stores occurs through channels formed by IP3 receptors (IP3Rs), and entry is mediated by a set of functionally heterogeneous but ubiquitous channels that are activated by the store depletion event per se. Transient receptor potential (TRP) proteins have been hypothesized to be structural components of Ca 2ϩ entry channels (4, 5) and to be activated by IP3R in response to IP3 or store depletion. However, neither has the presence of TRP in Ca 2ϩ entry channels been proven nor has the mechanism(s) by which the channels are activated been clearly elucidated. Indeed, the mechanism by which Ca 2ϩ entry channels are activated has received considerable attention, and arguments have been set forth (i) for activation by second messengers or mediators that include cGMP, IP3, diacylglycerol, a G protein, arachidonic acid derivatives, and a complex termed CIF (6-14), (ii) for translocation from internal pools with involvement of an exocytotic event (15, 16), and (iii) for shortrange physical coupling between the membrane delimiting the store and the plasma membrane (17, 18). The short-range physical-coupling model proposed that membrane Ca 2ϩ entry channels may be activated by the same protein that is responsible for store depletion, i.e., the IP3R (for details see ref. 1).The first functional evidence for a direct role of IP3R in Ca 2ϩ entry was obtained by Kiselyov et al. (19), who showed that Ca 2ϩ entry channels found in HEK cells expressing transfected TRP3 in stable form can be activated in inside-out membrane patches by addition of either IP3R-rich cerebellar microsomes or liposomes carrying recombinant IP3R protein truncated at its C terminus to inactivate its channel-forming capacity. However, this study did not determine whether the protein with which IP3R interacted was TRP3. Indeed, TRP was shown to cause changes in protein expression other than TRP, e.g., upregulation of IP3Rs, and attempts to coimmunoprecipitate IP3R and TRP3 failed (19). We now show that IP3R and TRP can be coimmunoprecipitated. We thus sought to identify interacting domains using in vitro protein:protein interaction tests and, if we found them, to test for their function. Such domains were identified and, upon expression in cells whose TRP and IP3R complement had not been manipulated, were found to modulate natural Ca 2ϩ entry stimulated by either a G protein-coupled pathway or store depletion. The data support a model in which store depletion-activated Ca 2ϩ entry is mediated by TRP-based channels that are activated by the IP3R. Materials and Methods

Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6 ؊/؊ smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6 ؊/؊ smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone.The TRP (transient receptor potential) family of ion channels is a growing group of structurally and evolutionarily related cation channels formed of several subfamilies that include the TRPC, TRPM, and TRPV classes of channels (6, 22). TRP-type ion channels are presumed to be homo-or heterotetramers (13,14), each spanning the plasma membrane six times. The founding members of this channel family are the insect TRP and TRPL channels, which are responsible for photoreceptor depolarization in response to light. Mammalian TRPCs (C stands for canonical or classical) (23, 32) are the closest mammalian structural relatives of insect TRPs. Among the TRPC channels, TRPC3, -6, and -7 are 75% identical and gated by signal transduction pathways that activate C-type phospholipases (3, 32) as well as by direct exposure to diacylglycerols (DAG) (15). TRPC3, -6, and -7 interact physically and, upon coexpression, coassemble to form functional channels (14). Expression of TRPC3 and TRPC7 in HEK 293 cells, but not of TRPC6, reveals constitutively active cation channels that are permeable not only to monovalent but also to divalent cations such as Ca 2ϩ , Ba 2ϩ , and Mn 2ϩ (7,23,33). In contrast to members of other TRP families, the functional importance of most members of the TRPC subfamily is still poorly understood. A TRPC channel for which considerable evidence has accumulated for a specific role is TRPC6, which has been proposed to regulate smooth muscle function. The TRPC6 mRNA was originally isolated from mouse brain and was also identified in lung cells (4). By comparative biophysical characterization and gene suppression using antisense oligonucleot...

Agonist-induced Ca 2؉ entry via store-operated Ca 2؉ (SOC) channels is suggested to regulate a wide variety of cellular functions, including salivary gland fluid secretion. However, the molecular components of these channels and their physiological function(s) are largely unknown. Here we report that attenuation of SOC current underlies salivary gland dysfunction in mice lacking transient receptor potential 1 (TRPC1). Neurotransmitter-regulated salivary gland fluid secretion in TRPC1-deficient TRPC1(؊/؊) mice was severely decreased (by 70%). Further, agonist-and thapsigargin-stimulated SOC channel activity was significantly reduced in salivary gland acinar cells isolated from TRPC1(؊/؊) mice. Deletion of TRPC1 also eliminated sustained Ca 2؉ -dependent potassium channel activity, which depends on Ca 2؉ entry and is required for fluid secretion. Expression of key proteins involved in fluid secretion and Ca 2؉ signaling, including STIM1 and other TRPC channels, was not altered. Together, these data demonstrate that reduced SOC entry accounts for the severe loss of salivary gland fluid secretion in TRPC1(؊/؊) mice. Thus, TRPC1 is a critical component of the SOC channel in salivary gland acinar cells and is essential for neurotransmitter-regulation of fluid secretion. transient receptor potential ͉ canonical ͉ Ca 2ϩ entry ͉ acinar cells ͉ muscarinic receptor

Among the classical transient receptor potential (TRPC) subfamily, TRPC1 is described as a mechanosensitive and store-operated channel proposed to be activated by hypoosmotic cell swelling and positive pipette pressure as well as regulated by the filling status of intracellular Ca(2+) stores. However, evidence for a physiological role of TRPC1 may most compellingly be obtained by the analysis of a TRPC1-deficient mouse model. Therefore, we have developed and analyzed TRPC1(-/-) mice. Pressure-induced constriction of cerebral arteries was not impaired in TRPC1(-/-) mice. Smooth muscle cells from cerebral arteries activated by hypoosmotic swelling and positive pipette pressure showed no significant differences in cation currents compared to wild-type cells. Moreover, smooth muscle cells of TRPC1(-/-) mice isolated from thoracic aortas and cerebral arteries showed no change in store-operated cation influx induced by thapsigargin, inositol-1,4,5 trisphosphate, and cyclopiazonic acid compared to cells from wild-type mice. In contrast to these results, small interference RNAs decreasing the expression of stromal interaction molecule 1 (STIM1) inhibited thapsigargin-induced store-operated cation influx, demonstrating that STIM1 and TRPC1 are mutually independent. These findings also imply that, as opposed to current concepts, TRPC1 is not an obligatory component of store-operated and stretch-activated ion channel complexes in vascular smooth muscle cells.