Highlights d Nephron-specific Tulp3 knockouts develop cystic kidneys without disrupting cilia d Cystogenesis is intermediate between that caused by loss of polycystin-1 or cilia d Polycystin-2 and Arl13b are reduced in cilia of knockout collecting duct cells d Arl13b is the likely polycystin-independent ciliary factor repressing cystogenesis
The T-box transcription factor Tbx1 has been implicated in DiGeorge syndrome, the most frequent syndrome due to a chromosomal deletion. Gene inactivation of Tbx1 in mice results in craniofacial and branchial arch defects, including myogenic defects in the first and second branchial arches. A T-box binding site has been identified in the Xenopus Myf5 promoter, and in other species, T-box genes have been implicated in myogenic fate. Here we analyze Tbx1 expression in the developing chick embryo relating its expression to the onset of myogenic differentiation and cellular fate within the craniofacial mesoderm. We show that Tbx1 is expressed before capsulin, the first known marker of branchial arch 1 and 2 muscles. We also show that, as in the mouse, Tbx1 is expressed in endothelial cells, another mesodermal derivative, and, therefore, Tbx1 alone cannot specify the myogenic lineage. In addition, Tbx1 expression was identified in both chick and mouse limb myogenic cells, initially being restricted to the dorsal muscle mass, but in contrast, to the head, here Tbx1 is expressed after the onset of myogenic commitment. Functional studies revealed that loss of Tbx1 function reduces the number of myocytes in the head and limb, whereas increasing Tbx1 activity has the converse effect. Finally, analysis of the Tbx1-mesoderm-specific knockout mouse demonstrated the cell autonomous requirement for Tbx1 during myocyte development in the cranial mesoderm. Developmental Dynamics 236:353-363, 2007.
Mutations in the polycystin proteins, PC-1 and PC-2, result in autosomal dominant polycystic kidney disease (ADPKD) and ultimately renal failure. PC-1 and PC-2 enrich on primary cilia, where they are thought to form a heteromeric ion channel complex. However, a functional understanding of the putative PC-1/PC-2 polycystin complex is lacking due to technical hurdles in reliably measuring its activity. Here, we successfully reconstitute the PC-1/PC-2 complex in the plasma membrane of mammalian cells and show that it functions as an outwardly rectifying channel. Using both reconstituted and ciliary polycystin channels, we further show that a soluble fragment generated from the N-terminal extracellular domain of PC-1 functions as an intrinsic agonist that is necessary and sufficient for channel activation. We thus propose that autoproteolytic cleavage of the N-terminus of PC-1, a hotspot for ADPKD mutations, produces a soluble ligand in vivo. These findings establish a mechanistic framework for understanding the role of PC-1/PC-2 heteromers in ADPKD and suggest new therapeutic strategies that would expand upon the limited symptomatic treatments currently available for this progressive, terminal disease.
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2 gene, encoding the polycystic kidney disease protein polycystin‐1 and the transient receptor potential channel polycystin‐2 (also known as TRPP2), respectively. Polycystin‐1 and polycystin‐2 form a receptor–ion channel complex located in primary cilia. The function of this complex, especially the role of polycystin‐1, is largely unknown due to the lack of a reliable functional assay. In this study, we dissect the role of polycystin‐1 by directly recording currents mediated by a gain‐of‐function (GOF) polycystin‐1/polycystin‐2 channel. Our data show that this channel has distinct properties from that of the homomeric polycystin‐2 channel. The polycystin‐1 subunit directly contributes to the channel pore, and its eleven transmembrane domains are sufficient for its channel function. We also show that the cleavage of polycystin‐1 at the N‐terminal G protein‐coupled receptor proteolysis site is not required for the activity of the GOF polycystin‐1/polycystin‐2 channel. These results demonstrate the ion channel function of polycystin‐1 in the polycystin‐1/polycystin‐2 complex, enriching our understanding of this channel and its role in ADPKD.
The human PKD2 locus encodes Polycystin-2 (PC2), a TRPP channel that localises to several distinct cellular compartments, including the cilium. PKD2 mutations cause Autosomal Dominant Polycystic Kidney Disease (ADPKD) and affect many cellular pathways. Data underlining the importance of ciliary PC2 localisation in preventing PKD are limited because PC2 function is ablated throughout the cell in existing model systems. Here, we dissect the ciliary role of PC2 by analysing mice carrying a non-ciliary localising, yet channel-functional, PC2 mutation. Mutants develop embryonic renal cysts that appear indistinguishable from mice completely lacking PC2. Despite not entering the cilium in mutant cells, mutant PC2 accumulates at the ciliary base, forming a ring pattern consistent with distal appendage localisation. This suggests a two-step model of ciliary entry; PC2 first traffics to the cilium base before TOP domain dependent entry. Our results suggest that PC2 localisation to the cilium is necessary to prevent PKD.
The role of mechanical forces driving kidney epithelial fluid transport and morphogenesis in kidney diseases is unclear. Here, using a microfluidic platform to recapitulate fluid transport activity of kidney cells, we report that renal epithelial cells can actively generate hydraulic pressure gradients across the epithelium. The fluidic flux declines with increasing hydraulic pressure until a stall pressure, in a manner similar to mechanical fluid pumps. For normal human kidney cells, the fluidic flux is from apical to basal, and the pressure is higher on the basal side. For human Autosomal Dominant Polycystic Kidney Disease cells, the fluidic flux is reversed from basal to apical. Molecular and proteomic studies reveal that renal epithelial cells are sensitive to hydraulic pressure gradients, changing gene expression profiles and spatial arrangements of ion exchangers and the cytoskeleton in different pressure conditions. These results implicate mechanical force and hydraulic pressure as important variables during kidney function and morphological change, and provide insights into pathophysiological mechanisms underlying the development and transduction of hydraulic pressure gradients.
Primary cilia play counterregulatory roles in cystogenesis—they inhibit cyst formation in the normal renal tubule but promote cyst growth when the function of polycystins is impaired. Key upstream cilia-specific signals and components involved in driving cystogenesis have remained elusive. Recent studies of the tubby family protein, Tubby-like protein 3 (TULP3), have provided new insights into the cilia-localized mechanisms that determine cyst growth. TULP3 is a key adapter of the intraflagellar transport complex A (IFT-A) in the trafficking of multiple proteins specifically into the ciliary membrane. Loss of TULP3 results in the selective exclusion of its cargoes from cilia without affecting their extraciliary pools and without disrupting cilia or IFT-A complex integrity. Epistasis analyses have indicated that TULP3 inhibits cystogenesis independently of the polycystins during kidney development but promotes cystogenesis in adults when polycystins are lacking. In this review, we discuss the current model of the cilia-dependent cyst activation (CDCA) mechanism in autosomal dominant polycystic kidney disease (ADPKD) and consider the possible roles of ciliary and extraciliary polycystins in regulating CDCA. We then describe the limitations of this model in not fully accounting for how cilia single knockouts cause significant cystic changes either in the presence or absence of polycystins. Based on available data from TULP3/IFT-A-mediated differential regulation of cystogenesis in kidneys with deletion of polycystins either during development or in adulthood, we hypothesize the existence of cilia-localized components of CDCA (cCDCA) and cilia-localized cyst inhibition (CLCI) signals. We develop the criteria for cCDCA/CLCI signals and discuss potential TULP3 cargoes as possible cilia-localized components that determine cystogenesis in kidneys during development and in adult mice.
Ketamine, a widely used anesthetic, is currently under renewed inquiry for its rapid and sustained antidepressant effects at sub anesthetic doses. Ketamine exerts its effects on the central nervous system (CNS) by blocking open Nmethyl-D-aspartate (NMDA) receptors with IC 50 = 1.0 uM. This overall effect is modulated regionally and temporally by dynamic variations in extracellular pH at synapses. An increase in extracellular proton concentrations decrease NMDA receptor open probability (Po) with IC 50 = 7.0 in an isoform dependent manner, and protonated forms of ketamine are more potent blockers of NMDA receptors. In an effort to separate the effects of proton inhibition and ketamine block on channel activity, we used GluN1-1b containing NMDA receptors with reduced proton sensitivity (IC 50 = 6.7) expressed in HEK293 cells and quantified the receptors' activation mechanism with kinetic modelling from on-cell single channel recordings. With this approach, we estimated rate constants for channel gating and for ketamine association and dissociation at pH 6.8, 7.2, and 7.6. The activation mechanism of protonated GluN1-1a and GluN1-1b isoforms were indistinguishable. As expected, ketamine reduced the channel mean open time with no changes in unitary conductance. Furthermore, ketamine increased the mean closed time and reduced the number of open components. These results quantify the distinct effects of protons and ketamine on NMDA receptor activity, and serve to better understand the complex modulatory actions of pharmacologic interventions.
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