An ATP-regulated, inwardly rectifying potassium channel from rat kidney (ROMK)

Potassium channels exhibit a wide functional diversity making them well suited for their broad roles in renal (and other) cells [1, 2]. Potassium channels can be classified into two broad groups based on their functional/biophysical properties: the delayed or outward rectifiers that are activated by depolarizing potentials and the inward rectifiers that include the classical (strongly) inwardly rectifying K+ channel and the more weakly inwardly rectifying ATP-sensitive potassium (KATP) channels [1, 3–7]. The inward rectifiers are characterized by a lack of significant gating by voltage and by their ability to conduct potassium more readily in the inward than outward direction. The classical (strong) and KATP-type of inward rectifiers have been identified in a variety of excitable and nonexcitable cells. The strong inward rectifiers appear to function in maintaining the resting membrane potential and in regulating excitability (such as in cardiac muscle cells). ATP-sensitive potassium channels, on the other hand, open and close in response to cellular metabolic events and may serve important roles in some cells during ischemia. Renal KATP channels, while sharing many of the properties and characteristics of KATP channels found in other tissues (such as pancreatic β-cell and cardiac muscle cells [3]), lack sensitivity to TEA, have a much lower sensitivity to sulfonylureas (such as glyburide, a high affinity inhibitor of KATP channels found in heart and β-cells), and require higher (that is, mM) concentrations of ATP to inhibit channel activity [1, 7].In the kidney, the apical (K+ secretory) KATP channel serves a number of important roles in renal electrolyte transport [1]. In the thick ascending limb of Henle (TAL; both medullary, MTAL, and cortical, CTAL, segments; Fig. 1), KATP channels are the dominant conductance in apical plasma membranes and provide a crucial K+ efflux pathway for potassium entering cells via the apical Na+:K+:2Cl- cotransporter [1]. This recycling of potassium ensures that an adequate supply of luminal potassium is provided for efficient function of the Na+:K+:2Cl- cotransporter [1]. In addition, this channel mediates the apical component of a transcellular (basolateral-to-apical) current flow that returns to the basolateral side via the paracellular pathway predominantly as a sodium current [8]. This provides for one-half of the net transepithelial movement of sodium [9]. Two types of inwardly rectifying and ATP-sensitive K+ channels have been identified on apical membranes of TAL segments by patch clamp [10, 11]. Wang and coworkers [10] found a 20 to 30 pS K+ channel in rabbit TAL that had a high open probability (Po), was inhibited by ATP (mM) and not sensitive to TEA (referred to as the “low conductance” channel). On the other hand, a different KATP channel was identified on apical membranes of rat TAL by Greger and coworkers [11–13]; this channel also had a high Po and was ATP-sensitive but had a higher unitary conductance of ∼70 pS, was highly sensitive to reductions in cytosolic side pH (50% reduction in Po by a 0.2 pH unit decrease), and exhibited sensitivity to quinine or quinidine, TEA and Ca2+ (referred to as the “intermediate conductance” channel). Recently, Wang found both the low (∼30 pS) and intermediate (∼72 pS) conductance KATP channels in the same patches of rat TAL apical membranes [14]. He also confirmed that the intermediate conductance KATP channel is sensitive to quinidine and acidic pH while the low conductance channel is insensitive to quinidine. In addition, the low, but not the intermediate, conductance channel is inhibited by high (∼250 µM) gliburide. These studies demonstrate that there are two distinct channel types in apical membranes of TAL and that these channels can be distinguished by single channel conductances and their sensitivities to channel inhibitors.A functionally similar, if not identical, low conductance, inwardly rectifying KATP channel has been identified in apical membranes of principal cells in the cortical collecting duct (CCD) where it mediates K+ secretion into urine (Fig. 1) [1, 7, 15, 16]. The KATP channel in rat principal cells is dually regulated by ATP: high MgATP concentrations reversibly block channel activity (K1/2 = 0.6 to 1.0 mM) while lower concentrations of MgATP are required to maintain channel activity [1, 7, 17, 18]. The mechanism for ATP-mediated block of the principal cell KATP channel is unclear at present but may represent direct binding of nucleotide to the channel itself with a resulting change in channel conformation to the closed state and/or to altering the activity of the channel by regulating the phosphorylation of the channel itself, or some other protein involved to modulating channel activity. The stimulatory effect of low ATP concentration, however, clearly relates to regulation of channel activity by phosphorylation-dephosphorylation processes [18]: (i) channel activity rapidly diminishes (run-down) on patch excision unless the cytosolic face is exposed to low concentrations of MgATP; (ii) generally the catalytic subunit of cAMP-dependent protein kinase, PKA, is also required for channel maintenance and PKA, and together with MgATP can restore channel activity after run-down; (iii) non-hydrolyzable ATP analogues cannot maintain or restore channel activity; (iv) in patches in which channel activity is maintained by MgATP alone, the PKA inhibitor (PKI) reversibly reduces channel activity, providing evidence for an important role for endogenous PKA; and (v) PKC reversibly inhibits channel activity and antagonizes the stimulatory effect of PKA, a process that is Ca2+-dependent [19]. In further studies Wang and Giebisch [17, 18] demonstrated that the ratio of ATP to ADP and cell pH are also important regulators of the small conductance KATP channel in the apical membranes of principal cells. KATP channel activity in rat principal cells is also inhibited by activation of protein kinase C [19] or calcium-calmodulin-dependent kinase II [20] or by arachidonic acid [21].Much less is known about the regulation of the apical KATP channels in the TAL than in the CCD; however, we previously suggested that AVP (presumably cyclic AMP-dependent activation of PKA and subsequent phosphorylation of the channel or an associated regulatory protein) activated the K+ conductance of the apical membrane in mouse MTAL [22, 23]. Reeves and coworkers have provided more direct evidence for this [24]. They showed that Ba2+-sensitive, voltage-dependent 86Rb+ influx in membrane vesicles from rabbit outer medulla was activated by cAMP-dependent protein kinase. Wang [14] has confirmed this effect of cAMP-PKA by showing activation of the low conductance KATP channel in cell attached patches by AVP or cAMP and in excised patches by the catalytic subunit of PKA.Finally, it should be noted that large conductance (maxi-K+), Ca2+-activated K+ channels have been identified in apical membranes of both TAL [25, 26] and CCD [1, 27–29]. These voltage-gated channels are normally quiescent but can be activated by µM cytosolic Ca2+, are inhibited by TEA (more sensitive to TEA than the intermediate conductance KATP channel), and are insensitive to ATP. Since K+(Rb+) secretion and the transepithelial voltage in the CCD are not blocked by luminal TEA [16, 28], it is generally thought that this apical maxi-K+ channel is not directly involved in K+ secretion by this nephron segment. The maxi-K+ channel may function, however, as a K+ efflux pathway during cell swelling [1, 7, 30]

Clustering of Unhealthy Behaviors in the Aerobics Center Longitudinal Study

Background Clustering of unhealthy behaviors has been reported in previous studies; however the link with all-cause mortality and differences between those with and without chronic disease requires further investigation. Objectives To observe the clustering effects of unhealthy diet, fitness, smoking, and excessive alcohol consumption in adults with and without chronic disease and to assess all-cause mortality risk according to the clustering of unhealthy behaviors. Methods Participants were 13,621 adults (aged 20–84) from the Aerobics Center Longitudinal Study. Four health behaviors were observed (diet, fitness, smoking, and drinking). Baseline characteristics of the study population and bivariate relations between pairs of the health behaviors were evaluated separately for those with and without chronic disease using cross-tabulation and a chi-square test. The odds of partaking in unhealthy behaviors were also calculated. Latent class analysis (LCA) was used to assess clustering. Cox regression was used to assess the relationship between the behaviors and mortality. Results The four health behaviors were related to each other. LCA results suggested that two classes existed. Participants in class 1 had a higher probability of partaking in each of the four unhealthy behaviors than participants in class 2. No differences in health behavior clustering were found between participants with and without chronic disease. Mortality risk increased relative to the number of unhealthy behaviors participants engaged in. Conclusion Unhealthy behaviors cluster together irrespective of chronic disease status. Such findings suggest that multi-behavioral intervention strategies can be similar in those with and without chronic disease

Multiple Multilocus DNA Barcodes from the Plastid Genome Discriminate Plant Species Equally Well

A universal barcode system for land plants would be a valuable resource, with potential utility in fields as diverse as ecology, floristics, law enforcement and industry. However, the application of plant barcoding has been constrained by a lack of consensus regarding the most variable and technically practical DNA region(s). We compared eight candidate plant barcoding regions from the plastome and one from the mitochondrial genome for how well they discriminated the monophyly of 92 species in 32 diverse genera of land plants (N = 251 samples). The plastid markers comprise portions of five coding (rpoB, rpoC1, rbcL, matK and 23S rDNA) and three non-coding (trnH-psbA, atpF–atpH, and psbK–psbI) loci. Our survey included several taxonomically complex groups, and in all cases we examined multiple populations and species. The regions differed in their ability to discriminate species, and in ease of retrieval, in terms of amplification and sequencing success. Single locus resolution ranged from 7% (23S rDNA) to 59% (trnH-psbA) of species with well-supported monophyly. Sequence recovery rates were related primarily to amplification success (85–100% for plastid loci), with matK requiring the greatest effort to achieve reasonable recovery (88% using 10 primer pairs). Several loci (matK, psbK–psbI, trnH-psbA) were problematic for generating fully bidirectional sequences. Setting aside technical issues related to amplification and sequencing, combining the more variable plastid markers provided clear benefits for resolving species, although with diminishing returns, as all combinations assessed using four to seven regions had only marginally different success rates (69–71%; values that were approached by several two- and three-region combinations). This performance plateau may indicate fundamental upper limits on the precision of species discrimination that is possible with DNA barcoding systems that include moderate numbers of plastid markers. Resolution to the contentious debate on plant barcoding should therefore involve increased attention to practical issues related to the ease of sequence recovery, global alignability, and marker redundancy in multilocus plant DNA barcoding systems

DNA Barcoding Bromeliaceae: Achievements and Pitfalls

<div><h3>Background</h3><p>DNA barcoding has been successfully established in animals as a tool for organismal identification and taxonomic clarification. Slower nucleotide substitution rates in plant genomes have made the selection of a DNA barcode for land plants a much more difficult task. The Plant Working Group of the Consortium for the Barcode of Life (CBOL) recommended the two-marker combination <em>rbcL</em>/<em>matK</em> as a pragmatic solution to a complex trade-off between universality, sequence quality, discrimination, and cost.</p> <h3>Methodology/Principal Findings</h3><p>It is expected that a system based on any one, or a small number of plastid genes will fail within certain taxonomic groups with low amounts of plastid variation, while performing well in others. We tested the effectiveness of the proposed CBOL Plant Working Group barcoding <em>markers</em> for land plants in identifying 46 bromeliad species, a group rich in endemic species from the endangered Brazilian Atlantic Rainforest. Although we obtained high quality sequences with the suggested primers, species discrimination in our data set was only 43.48%. Addition of a third marker, <em>trnH–psbA</em>, did not show significant improvement. This species identification failure in Bromeliaceaecould also be seen in the analysis of the GenBank's <em>matK</em> data set. Bromeliaceae's sequence divergence was almost three times lower than the observed for Asteraceae and Orchidaceae. This low variation rate also resulted in poorly resolved tree topologies. Among the three Bromeliaceae subfamilies sampled, Tillandsioideae was the only one recovered as a monophyletic group with high bootstrap value (98.6%). Species paraphyly was a common feature in our sampling.</p> <h3>Conclusions/Significance</h3><p>Our results show that although DNA barcoding is an important tool for biodiversity assessment, it tends to fail in taxonomy complicated and recently diverged plant groups, such as Bromeliaceae. Additional research might be needed to develop markers capable to discriminate species in these complex botanical groups.</p> </div

Remodeling Lipid Metabolism and Improving Insulin Responsiveness in Human Primary Myotubes

OBJECTIVE: Disturbances in lipid metabolism are strongly associated with insulin resistance and type 2 diabetes (T2D). We hypothesized that activation of cAMP/PKA and calcium signaling pathways in cultured human myotubes would provide further insight into regulation of lipid storage, lipolysis, lipid oxidation and insulin responsiveness. METHODS: Human myoblasts were isolated from vastus lateralis, purified, cultured and differentiated into myotubes. All cells were incubated with palmitate during differentiation. Treatment cells were pulsed 1 hour each day with forskolin and ionomycin (PFI) during the final 3 days of differentiation to activate the cAMP/PKA and calcium signaling pathways. Control cells were not pulsed (control). Mitochondrial content, (14)C lipid oxidation and storage were measured, as well as lipolysis and insulin-stimulated glycogen storage. Myotubes were stained for lipids and gene expression measured. RESULTS: PFI increased oxidation of oleate and palmitate to CO(2) (p<0.001), isoproterenol-stimulated lipolysis (p = 0.01), triacylglycerol (TAG) storage (p<0.05) and mitochondrial DNA copy number (p = 0.01) and related enzyme activities. Candidate gene and microarray analysis revealed increased expression of genes involved in lipolysis, TAG synthesis and mitochondrial biogenesis. PFI increased the organization of lipid droplets along the myofibrillar apparatus. These changes in lipid metabolism were associated with an increase in insulin-mediated glycogen storage (p<0.001). CONCLUSIONS: Activation of cAMP/PKA and calcium signaling pathways in myotubes induces a remodeling of lipid droplets and functional changes in lipid metabolism. These results provide a novel pharmacological approach to promote lipid metabolism and improve insulin responsiveness in myotubes, which may be of therapeutic importance for obesity and type 2 diabetes

Ethnobotany genomics - discovery and innovation in a new era of exploratory research

We present here the first use of DNA barcoding in a new approach to ethnobotany we coined "ethnobotany genomics". This new approach is founded on the concept of 'assemblage' of biodiversity knowledge, which includes a coming together of different ways of knowing and valorizing species variation in a novel approach seeking to add value to both traditional knowledge (TK) and scientific knowledge (SK). We employed contemporary genomic technology, DNA barcoding, as an important tool for identifying cryptic species, which were already recognized ethnotaxa using the TK classification systems of local cultures in the Velliangiri Hills of India. This research is based on several case studies in our lab, which define an approach to that is poised to evolve quickly with the advent of new ideas and technology. Our results show that DNA barcoding validated several new cryptic plant species to science that were previously recognized by TK classifications of the Irulas and Malasars, and were lumped using SK classification. The contribution of the local aboriginal knowledge concerning plant diversity and utility in India is considerable; our study presents new ethnomedicine to science. Ethnobotany genomics can also be used to determine the distribution of rare species and their ecological requirements, including traditional ecological knowledge so that conservation strategies can be implemented. This is aligned with the Convention on Biological Diversity that was signed by over 150 nations, and thus the world's complex array of human-natural-technological relationships has effectively been re-organized

Identification of β-Secretase (BACE1) Substrates Using Quantitative Proteomics

β-site APP cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease with a lumenal active site that sheds the ectodomains of membrane proteins through juxtamembrane proteolysis. BACE1 has been studied principally for its role in Alzheimer's disease as the β-secretase responsible for generating the amyloid-β protein. Emerging evidence from mouse models has identified the importance of BACE1 in myelination and cognitive performance. However, the substrates that BACE1 processes to regulate these functions are unknown, and to date only a few β-secretase substrates have been identified through candidate-based studies. Using an unbiased approach to substrate identification, we performed quantitative proteomic analysis of two human epithelial cell lines stably expressing BACE1 and identified 68 putative β-secretase substrates, a number of which we validated in a cell culture system. The vast majority were of type I transmembrane topology, although one was type II and three were GPI-linked proteins. Intriguingly, a preponderance of these proteins are involved in contact-dependent intercellular communication or serve as receptors and have recognized roles in the nervous system and other organs. No consistent sequence motif predicting BACE1 cleavage was identified in substrates versus non-substrates. These findings expand our understanding of the proteins and cellular processes that BACE1 may regulate, and suggest possible mechanisms of toxicity arising from chronic BACE1 inhibition

Towards the new Thematic Core Service Tsunami within the EPOS Research Infrastructure

Tsunamis constitute a significant hazard for European coastal populations, and the impact of tsunami events worldwide can extend well beyond the coastal regions directly affected. Understanding the complex mechanisms of tsunami generation, propagation, and inundation, as well as managing the tsunami risk, requires multidisciplinary research and infrastructures that cross national boundaries. Recent decades have seen both great advances in tsunami science and consolidation of the European tsunami research community. A recurring theme has been the need for a sustainable platform for coordinated tsunami community activities and a hub for tsunami services. Following about three years of preparation, in July 2021, the European tsunami community attained the status of Candidate Thematic Core Service (cTCS) within the European Plate Observing System (EPOS) Research Infrastructure. Within a transition period of three years, the Tsunami candidate TCS is anticipated to develop into a fully operational EPOS TCS. We here outline the path taken to reach this point, and the envisaged form of the future EPOS TCS Tsunami. Our cTCS is planned to be organised within four thematic pillars: (1) Support to Tsunami Service Providers, (2) Tsunami Data, (3) Numerical Models, and (4) Hazard and Risk Products. We outline how identified needs in tsunami science and tsunami risk mitigation will be addressed within this structure and how participation within EPOS will become an integration point for community development.publishedVersio

Towards the new Thematic Core Service Tsunami within the EPOS Research Infrastructure

publishedVersio