The activation mechanism of the classical transient receptor potential channels TRPC4 and -5 via the G q/11 protein-phospholipase C (PLC) signaling pathway has remained elusive so far. In contrast to all other TRPC channels, the PLC product diacylglycerol (DAG) is not sufficient for channel activation, whereas TRPC4/5 channel activity is potentiated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) depletion. As a characteristic structural feature, TRPC4/5 channels contain a C-terminal PDZ-binding motif allowing for binding of the scaffolding proteins Na+ exchanger regulatory factor (NHERF) 1 and 2. PKC inhibition or the exchange of threonine for alanine in the C-terminal PDZ-binding motif conferred DAG sensitivity to the channel. Altogether, we present a DAG-mediated activation mechanism for TRPC4/5 channels tightly regulated by NHERF1/2 interaction. PIP 2 depletion evokes a C-terminal conformational change of TRPC5 proteins leading to dynamic dissociation of NHERF1/2 from the C terminus of TRPC5 as a prerequisite for DAG sensitivity. We show that NHERF proteins are direct regulators of ion channel activity and that DAG sensitivity is a distinctive hallmark of TRPC channels.and -5 channels are members of the classical transient receptor potential cation (TRPC) family of nonselective, calcium permeable receptor-operated cation channels. They are widely expressed in many tissues, including brain, kidney, and the vascular system. High expression levels are found in the central nervous system where TRPC4 and -5 are involved in amygdala function and fear-related behavior (1, 2), seizure, and excitotoxicity (3). Furthermore, TRPC5 channels are implicated in neuronal depolarization and bursting during epiletiform seizures (4) and regulate hippocampal neurite length and growth cone morphology (5). In the kidney, TRPC5 channels are proposed to be protective against renal failure (6). TRPC channels are usually activated by G q/11 proteincoupled receptors via phospholipase C (PLC) activation resulting in cleavage of phosphatidylinositol-3,4-bisphosphate (PIP 2 ) into the second messengers inositol-1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG). DAG is known to activate TRPC2, -3, -6, and -7 (7-9) channels, whereas TRPC4 and -5 are supposed to be insensitive to the PLC product DAG (8) and are even inhibited by DAG or its membrane-permeable analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) (10). DAG-mediated TRPC5 channel inhibition was shown to be PKC dependent (10). Furthermore, TRPC4 and -5 channels can be activated by depleting PIP 2 (11, 12), contrary to TRPC6 and -7 channels, which are inhibited by PIP 2 depletion (13). However, there are first hints to show that endogenously expressed TRPC5 channels might be DAG sensitive (14) but mechanistic insight is lacking so far.A noteworthy structural difference between TRPC4 and -5 and the established DAG-sensitive TRPC3, -6, and -7 channels is the PDZbinding motif VTTLR in the C termini of TRPC4 and -5 channels (15-17) as a structural basis of the interaction with Na + /H + e...

Specific biological roles of the classical transient receptor potential channel 1 (TRPC1) are still largely elusive. To investigate the function of TRPC1 proteins in cell physiology, we studied heterologously expressed TRPC1 channels and found that recombinant TRPC1 subunits do not form functional homomeric channels. Instead, by electrophysiological analysis TRPC1 was shown to form functional heteromeric, receptoroperated channel complexes with TRPC3, -4, -5, -6, and -7 indicating that TRPC1 proteins can co-assemble with all members of the TRPC subfamily. In all TRPC1-containing heteromers, TRPC1 subunits significantly decreased calcium permeation. The exchange of select amino acids in the putative pore-forming region of TRPC1 further reduced calcium permeability, suggesting that TRPC1 subunits contribute to the channel pore. In immortalized immature gonadotropin-releasing hormone neurons endogenously expressing TRPC1, -2, -5, and -6, down-regulation of TRPC1 resulted in increased calcium permeability and elevated basal cytosolic calcium concentrations. We did not observe any involvement of TRPC1 in storeoperated cation influx. Notably, TRPC1 suppressed the migration of gonadotropin-releasing hormone neurons without affecting cell proliferation. Conversely, in TRPC1 knockdown neurons, specific migratory properties like distance covered, locomotion speed, and directionality were increased. These findings suggest a novel regulatory mechanism relying on the expression of TRPC1 and the subsequent formation of heteromeric TRPC channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration.The classical transient receptor potential (TRPC) 2 channel subfamily comprises seven members (TRPC1-7) that are regarded as non-selective, calcium-permeable cation channels involved in a wide range of physiological events that require calcium (Ca 2ϩ ) signaling. To date, it is broadly accepted that the general activation mechanism of TRPC channels is contingent upon receptor-mediated phospholipase C activation independent of protein kinase C activity and the depletion of internal calcium stores (1). However, channel activation subsequent to store depletion is also discussed for some TRPC family members (summarized by Ref. 2). TRPC channels are widely expressed in different mammalian tissues like vascular smooth muscle, lung, kidney, and brain, and they have been identified to participate in central cell physiological processes (3). In the nervous system, for example, TRPC channels are involved in neuronal development, proliferation, and differentiation (4, 5), and a growing body of evidence indicates that TRPC channels are involved in neurological diseases (6).For TRPC1 channels, an involvement in stretch-induced (7) and in store-operated calcium (SOC) influx is discussed (8 -10). Previous investigations of TRPC1 gene-deficient mice indicated that TRPC1 was neither involved in store-operated cation influx in vascular smooth muscle cells and in platelets (11, 12) nor in pressure-induced cation influx (11...

BackgroundThe transepithelial transport of electrolytes, solutes, and water in the kidney is a well-orchestrated process involving numerous membrane transport systems. Basolateral potassium channels in tubular cells not only mediate potassium recycling for proper Na+,K+-ATPase function but are also involved in potassium and pH sensing. Genetic defects in KCNJ10 cause EAST/SeSAME syndrome, characterized by renal salt wasting with hypokalemic alkalosis associated with epilepsy, ataxia, and sensorineural deafness.MethodsA candidate gene approach and whole-exome sequencing determined the underlying genetic defect in eight patients with a novel disease phenotype comprising a hypokalemic tubulopathy with renal salt wasting, disturbed acid-base homeostasis, and sensorineural deafness. Electrophysiologic studies and surface expression experiments investigated the functional consequences of newly identified gene variants.ResultsWe identified mutations in the KCNJ16 gene encoding KCNJ16, which along with KCNJ15 and KCNJ10, constitutes the major basolateral potassium channel of the proximal and distal tubules, respectively. Coexpression of mutant KCNJ16 together with KCNJ15 or KCNJ10 in Xenopus oocytes significantly reduced currents.ConclusionsBiallelic variants in KCNJ16 were identified in patients with a novel disease phenotype comprising a variable proximal and distal tubulopathy associated with deafness. Variants affect the function of heteromeric potassium channels, disturbing proximal tubular bicarbonate handling as well as distal tubular salt reabsorption.

Myogenic vasoconstriction is an inherent property of vascular smooth muscle cells (VSMCs) of resistance arteries harboring ill-defined mechanosensing and mechanotransducing elements. G protein-coupled receptors (GPCRs) are discussed as mechanosensors in VSMCs. In this study, we sought to identify and characterize the role and impact of GPCRs on myogenic vasoconstriction. Thus, we analyzed mRNA expression levels of GPCRs in resistance versus preceding conduit arteries revealing a significant enrichment of several GPCRs in resistance vessels. Selective pharmacological blockade of the highly expressed GPCRs in isolated murine mesenteric arteries ex vivo decreased myogenic vasoconstriction. In particular, candesartan and losartan most prominently suppressed myogenic tone, suggesting that AT1 receptors play a central role in myogenic vasoconstriction. Analyzing angiotensinogen(-/-) mice, a relevant contribution of locally produced angiotensin II to myogenic tone could be excluded. Investigation of AT1A (-/-) and AT1B (-/-) murine mesenteric arteries revealed that AT1B, but not AT1A, receptors are key components of myogenic regulation. This notion was supported by examining fura-2-loaded isolated AT1A (-/-) and AT1B (-/-) VSMCs. Our results indicate that in VSMCs, AT1B receptors are more mechanosensitive than AT1A receptors even at comparable receptor expression levels. Furthermore, we demonstrate that the mechanosensitivity of GPCRs is agonist-independent and positively correlates with receptor expression levels.

Podocytes are specialized, highly differentiated epithelial cells in the kidney glomerulus that are exposed to glomerular capillary pressure and possible increases in mechanical load. The proteins sensing mechanical forces in podocytes are unconfirmed, but the classic transient receptor potential channel 6 (TRPC6) interacting with the MEC-2 homolog podocin may form a mechanosensitive ion channel complex in podocytes. Here, we observed that podocytes respond to mechanical stimulation with increased intracellular calcium concentrations and increased inward cation currents. However, TRPC6-deficient podocytes responded in a manner similar to that of control podocytes, and mechanically induced currents were unaffected by genetic inactivation of TRPC1/3/6 or administration of the broad-range TRPC blocker SKF-96365. Instead, mechanically induced currents were significantly decreased by the specific P2X purinoceptor 4 (P2X 4 ) blocker 5-BDBD. Moreover, mechanical P2X 4 channel activation depended on cholesterol and podocin and was inhibited by stabilization of the actin cytoskeleton. Because P2X 4 channels are not intrinsically mechanosensitive, we investigated whether podocytes release ATP upon mechanical stimulation using a fluorometric approach. Indeed, mechanically induced ATP release from podocytes was observed. Furthermore, 5-BDBD attenuated mechanically induced reorganization of the actin cytoskeleton. Altogether, our findings reveal a TRPC channel-independent role of P2X 4 channels as mechanotransducers in podocytes.