Sodium permeability of a cloned small-conductance calcium-activated potassium channel

Small-conductance Ca2+-activated potassium channels (SK(Ca) channels) are heteromeric complexes of pore-forming main subunits and constitutively bound calmodulin. SK(Ca) channels in neuronal cells are activated by intracellular Ca2+ that increases during action potentials, and their ionic currents have been considered to underlie neuronal afterhyperpolarization. However, the ion selectivity of neuronal SK(Ca) channels has not been rigorously investigated. In this study, we determined the monovalent cation selectivity of a cloned rat SK(Ca) channel, rSK2, using heterologous expression and electrophysiological measurements. When extracellular K+ was replaced isotonically with Na+, ionic currents through rSK2 reversed at significantly more depolarized membrane potentials than the value expected for a Nernstian relationship for K+. We then determined the relative permeability of rSK2 for monovalent cations and compared them with those of the intermediate- and large-conductance Ca2+-activated K+ channels, IK(Ca) and BK(Ca) channels. The relative permeability of the rSK2 channel was determined as K+(1.0)>Rb+(0.80)>NH(4)+(0.19) approximately Cs+(0.19)>Li+(0.14)>Na+(0.12), indicating substantial permeability of small ions through the channel. Although a mutation near the selectivity filter mimicking other K+-selective channels influenced the size-selectivity for permeant ions, Na+ permeability of rSK2 channels was still retained. Since the reversal potential of endogenous SK(Ca) current is determined by Na+ permeability in a physiological ionic environment, the ion selectivity of native SK(Ca) channels should be reinvestigated and their in vivo roles may need to be restated.     

Sat1 is dispensable for active oxalate secretion in mouse duodenum

Mice deficient for the apical membrane oxalate transporter SLC26A6 develop hyperoxalemia, hyperoxaluria, and calcium oxalate stones due to a defect in intestinal oxalate secretion. However, the nature of the basolateral membrane oxalate transport process that operates in series with SLC26A6 to mediate active oxalate secretion in the intestine remains unknown. Sulfate anion transporter-1 (Sat1 or SLC26A1) is a basolateral membrane anion exchanger that mediates intestinal oxalate transport. Moreover, Sat1-deficient mice also have a phenotype of hyperoxalemia, hyperoxaluria, and calcium oxalate stones. We, therefore, tested the role of Sat1 in mouse duodenum, a tissue with Sat1 expression and SLC26A6-dependent oxalate secretion. Although the active secretory flux of oxalate across mouse duodenum was strongly inhibited (>90%) by addition of the disulfonic stilbene DIDS to the basolateral solution, secretion was unaffected by changes in medium concentrations of sulfate and bicarbonate, key substrates for Sat1-mediated anion exchange. Inhibition of intracellular bicarbonate production by acetazolamide and complete removal of bicarbonate from the buffer also produced no change in oxalate secretion. Finally, active oxalate secretion was not reduced in Sat1-null mice. We conclude that a DIDS-sensitive basolateral transporter is involved in mediating oxalate secretion across mouse duodenum, but Sat1 itself is dispensable for this process.     

Phosphorylation-independent regulation of the diguanylate cyclase WspR

Environmental signals that trigger bacterial pathogenesis and biofilm formation are mediated by changes in the level of cyclic dimeric guanosine monophosphate (c-di-GMP), a unique eubacterial second messenger. Tight regulation of cellular c-di-GMP concentration is governed by diguanylate cyclases and phosphodiesterases, which are responsible for its production and degradation, respectively. Here, we present the crystal structure of the diguanylate cyclase WspR, a conserved GGDEF domain-containing response regulator in Gram-negative bacteria, bound to c-di-GMP at an inhibitory site. Biochemical analyses revealed that feedback regulation involves the formation of at least three distinct oligomeric states. By switching from an active to a product-inhibited dimer via a tetrameric assembly, WspR utilizes a novel mechanism for modulation of its activity through oligomerization. Moreover, our data suggest that these enzymes can be activated by phosphodiesterases. Thus, in addition to the canonical pathways via phosphorylation of the regulatory domains, both product and enzyme concentration contribute to the coordination of c-di-GMP signaling. A structural comparison reveals resemblance of the oligomeric states to assemblies of GAF domains, widely used regulatory domains in signaling molecules conserved from archaea to mammals, suggesting a similar mechanism of regulation.     

Relationship of Behavioral Interactions during an Animal-assisted Intervention in Assisted Living to Health-related Outcomes

ABSTRACT

In recent research involving assisted-living residents with mild to moderate cognitive impairment, a structured 12-week Pet Assisted Living (PAL) intervention led to improved physical activity (PA) and mood. Receipt of intervention is important to the efficacy of behavioral interventions. We assessed the behavioral interactions (BIs: looking at, talking to, touching, giving treats to, brushing, and walking the dog) of residents during PAL intervention sessions and evaluated their relationships to changes in PA and mood. Residents with mild to moderate cognitive impairment in seven small assisted-living facilities were randomized by facility to the 12-week PAL or reminiscing interventions. Outcomes included PA and mood. PA was assessed monthly with 24-hour actigraph and Barthel Index, and mood was assessed with the Cornell Scale for Depression in Dementia. Residents of four facilities (n = 22) received the PAL intervention which included bi-weekly activities designed to encourage maintenance or improvement of PA and mood. During each PAL session, participants’ engagement in each BI was noted. Engagement in BIs varied between residents and over sessions. BIs were not correlated with degree of cognitive impairment. In linear mixed models, the more participants walked the dog the more their PA changed, and the more participants looked at the dog the more their moods changed (ps < 0.05). Participants varied in their engagement in BIs. Two specific BIs were related to improvements in PA and mood. Finer assessment of BIs during animal-assisted interventions may enable understanding of their relationships to specific outcomes.     

Sorting nexin 9 interacts with dynamin 1 and N-WASP and coordinates synaptic vesicle endocytosis

Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins, each of which contains a characteristic Phox homology domain. SNX9 is widely expressed and plays a role in clathrin-mediated endocytosis, but it is not known if it is present in neuronal cells. We report that SNX9 is expressed in the presynaptic compartment of cultured hippocampal neurons, where it binds to dynamin-1 and N-WASP. Overexpression of full-length SNX9 or a C-terminal truncated version caused severe defects in synaptic vesicle endocytosis during, as well as after, stimulation. Knockdown of SNX9 with short interfering RNA also reduced synaptic vesicle endocytosis, and the W39A mutation of SNX9 abolished the inhibitory effect of SNX9 on endocytosis. Rescue experiments showed that most of the effect of SNX9 on endocytosis results from its interaction with dynamin 1, although its interaction with N-WASP contributes in some degree. We further showed that SNX9 dimerizes through its C-terminal domain, suggesting that it may interact simultaneously with dynamin 1 and N-WASP. We propose that SNX9 interacts with dynamin-1 and N-WASP in presynaptic terminals, where it links actin dynamics and synaptic vesicle endocytosis.     

Strategy for comprehensive identification of post-translational modifications in cellular proteins, including low abundant modifications: application to glyceraldehyde-3-phosphate dehydrogenase

Post-translational modifications (PTMs) play key roles in the regulation of biological functions of proteins. Although some progress has been made in identifying several PTMs using existing approaches involving a combination of affinity-based enrichment and mass spectrometric analysis, comprehensive identification of PTMs remains a challenging problem in proteomics because of the dynamic complexities of PTMs in vivo and their low abundance. We describe here a strategy for rapid, efficient, and comprehensive identification of PTMs occurring in biological processes in vivo. It involves a selectively excluded mass screening analysis (SEMSA) of unmodified peptides during liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-q-TOF MS/MS) through replicated runs of a purified protein on two-dimensional gel. A precursor ion list of unmodified peptides with high mass intensities was obtained during the initial run followed by exclusion of these unmodified peptides in subsequent runs. The exclusion list can grow as long as replicate runs are iteratively performed. This enables the identifications of modified peptides with precursor ions of low intensities by MS/MS sequencing. Application of this approach in combination with the PTM search algorithm MODi to GAPDH protein in vivo modified by oxidative stress provides information on multiple protein modifications (19 types of modification on 42 sites) with >92% peptide coverage and the additional potential for finding novel modifications, such as transformation of Cys to Ser. On the basis of the information of precursor ion m/z, quantitative analysis of PTM was performed for identifying molecular changes in heterogeneous protein populations. Our results show that PTMs in mammalian systems in vivo are more complicated and heterogeneous than previously reported. We believe that this strategy has significant potential because it permits systematic characterization of multiple PTMs in functional proteomics.     

Differential expression of major genes involved in the biosynthesis of aliphatic glucosinolates in intergeneric Baemoochae (Brassicaceae) and its parents during development

Plant Molecular Biology - Thus study found the temporal and spatial relationship between production of aliphatic glucosinolate compounds and the expression profile of glucosinolate-related genes...