In animals, silicon is an abundant and differentially distributed trace element that is believed to play important biological functions. One would thus expect silicon concentrations in body fluids to be regulated by silicon transporters at the surface of many cell types. Curiously, however, and even though they exist in plants and algae, no such transporters have been identified to date in vertebrates. Here, we show for the first time that the human aquaglyceroporins, i.e., AQP3, AQP7, AQP9 and AQP10 can act as silicon transporters in both Xenopus laevis oocytes and HEK-293 cells. In particular, heterologously expressed AQP7, AQP9 and AQP10 are all able to induce robust, saturable, phloretin-sensitive silicon transport activity in the range that was observed for low silicon rice 1 (lsi1), a silicon transporter in plant. Furthermore, we show that the aquaglyceroporins appear as relevant silicon permeation pathways in both mice and humans based on 1) the kinetics of substrate transport, 2) their presence in tissues where silicon is presumed to play key roles and 3) their transcriptional responses to changes in dietary silicon. Taken together, our data provide new evidence that silicon is a potentially important biological element in animals and that its body distribution is regulated. They should open up original areas of investigations aimed at deciphering the true physiological role of silicon in vertebrates.

Silicon (Si) has long been known to play a major physiological and structural role in certain organisms, including diatoms, sponges, and many higher plants, leading to the recent identification of multiple proteins responsible for Si transport in a range of algal and plant species. In mammals, despite several convincing studies suggesting that silicon is an important factor in bone development and connective tissue health, there is a critical lack of understanding about the biochemical pathways that enable Si homeostasis. Here we report the identification of a mammalian efflux Si transporter, namely Slc34a2 (also termed NaPiIIb), a known sodium-phosphate cotransporter, which was upregulated in rat kidney following chronic dietary Si deprivation. Normal rat renal epithelium demonstrated punctate expression of Slc34a2, and when the protein was heterologously expressed in Xenopus laevis oocytes, Si efflux activity (i.e., movement of Si out of cells) was induced and was quantitatively similar to that induced by the known plant Si transporter OsLsi2 in the same expression system. Interestingly, Si efflux appeared saturable over time, but it did not vary as a function of extracellular [Formula: see text] or Na+ concentration, suggesting that Slc34a2 harbors a functionally independent transport site for Si operating in the reverse direction to the site for phosphate. Indeed, in rats with dietary Si depletion-induced upregulation of transporter expression, there was increased urinary phosphate excretion. This is the first evidence of an active Si transport protein in mammals and points towards an important role for Si in vertebrates and explains interactions between dietary phosphate and silicon.

Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.

The K(+)-Cl(-) cotransporter KCC2 is responsible for maintaining low Cl(-) concentration in neurons of the central nervous system (CNS), which is essential for postsynaptic inhibition through GABA(A) and glycine receptors. Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain. Recent reports indicate that enhancing KCC2 activity may be the favored therapeutic strategy to restore inhibition and normal function in pathological conditions involving impaired Cl(-) transport. We designed an assay for high-throughput screening that led to the identification of KCC2 activators that reduce intracellular chloride concentration ([Cl(-)]i). Optimization of a first-in-class arylmethylidine family of compounds resulted in a KCC2-selective analog (CLP257) that lowers [Cl(-)]i. CLP257 restored impaired Cl(-) transport in neurons with diminished KCC2 activity. The compound rescued KCC2 plasma membrane expression, renormalized stimulus-evoked responses in spinal nociceptive pathways sensitized after nerve injury and alleviated hypersensitivity in a rat model of neuropathic pain. Oral efficacy for analgesia equivalent to that of pregabalin but without motor impairment was achievable with a CLP257 prodrug. These results validate KCC2 as a druggable target for CNS diseases.

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

Inactivation of Kcc3 in a mixed 129/Sv×C57BL/6 mouse background has been previously found to increase systemic blood pressure (BP) through presumed neurogenic mechanisms. Yet, while this background is generally not considered ideal to investigate the cardiovascular system, KCC3 is also expressed in the arterial wall and proximal nephron. In the current study, the effects of Kcc3 ablation was investigated in a pure rather than mixed C57BL/6J background under regular- and high-salt diets to determine whether they could be mediated through vasculogenic and nephrogenic mechanisms. Aortas were also assessed for reactivity to pharmacological agents while isolated from the influence of sympathetic ganglia. This approach led to the identification of unforeseen abnormalities such as lower pulse pressure, heart rate, aortic reactivity and aortic wall thickness, but higher diastolic BP, left ventricular mass and urinary output in the absence of increased catecholamine levels. Salt loading also led systolic BP to be higher, but to no further changes in hemodynamic parameters. Importantly, aortic vascular smooth muscle cells and cardiomyocytes were both found to express KCC3 abundantly in heterozygous mice. Hence, Kcc3 inactivation in our model caused systemic vascular resistance and ventricular mass to increase while preventing extracellular fluid volume to accumulate. Given that it also affected the physiological properties of aortas in vitro, vasculogenic mechanisms could therefore account for a number of the hemodynamic abnormalities observed.

Atypical hemolytic uremic syndrome (aHUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Complement and coagulation gene variants have been associated with aHUS susceptibility. We assessed the diagnostic yield of a next-generation sequencing (NGS) panel in a large cohort of Canadian patients with suspected aHUS. Molecular testing was performed on peripheral blood DNA samples from 167 patients, collected between May 2019 and December 2021, using a clinically validated NGS pipeline. Coding exons with 20 base pairs of flanking intronic regions for 21 aHUS-associated or candidate genes were enriched using a custom hybridization protocol. All sequence and copy number variants were assessed and classified following American College of Medical Genetics guidelines. Molecular diagnostic results were reported for four variants in three individuals (1.8%). Twenty-seven variants of unknown significance were identified in 25 (15%) patients, and 34 unique variants in candidate genes were identified in 28 individuals. An illustrative patient case describing two genetic alterations in complement genes is presented, highlighting that variable expressivity and incomplete penetrance must be considered when interpreting genetic data in patients with complement-mediated disease, alongside the potential additive effects of genetic variants on aHUS pathophysiology. In this cohort of patients with suspected aHUS, using clinical pipelines for genetic testing and variant classification, pathogenic/likely pathogenic variants occurred in a very small percentage of patients. Our results highlight the ongoing challenges in variant classification following NGS panel testing in patients with suspected aHUS, alongside the need for clear testing guidance in the clinical setting. KEY MESSAGES: • Clinical molecular testing for disease associated genes in aHUS is challenging. • Challenges include patient selection criteria, test validation, and interpretation. • Most variants were of uncertain significance (31.7% of patients; VUS + candidates). • Their clinical significance may be elucidated as more evidence becomes available.  • Low molecular diagnostic rate (1.8%), perhaps due to strict classification criteria. • Case study identified two likely pathogenic variants; one each in MCP/CD46 and CFI.

We recently demonstrated that the aquaglyceroporins (AQGPs) could act as potent transporters for orthosilicic acid (H4SiO4). Although interesting, this finding raised the question of whether water and H4SiO4, the transportable form of Si, permeate AQGPs by interacting with the same region of the pore, especially in view of the difference in molecular radius between the two substrates. Here, our goal was to identify residues that endow the AQGPs with the ability to facilitate Si diffusion by examining the transport characteristics of mutants in which residues were interchanged between a water-permeable but Si-impermeable channel (aquaporin 1 [AQP1]) and a Si-permeable but water-impermeable channel (AQP10). Our results indicate that the composition of the arginine filter (XX/R), known to include three residues that play an important role in water transport, may also be involved in Si selectivity. Interchanging the identities of the nonarginine residues within this filter causes Si transport to increase by approximately sevenfold in AQP1 and to decrease by approximately threefold in AQP10, whereas water transport and channel expression remain unaffected. Our results further indicate that two additional residues in the AQP arginine filter may be involved in substrate selectivity: replacing one of the residues has a profound effect on water permeability, and replacing the other has a profound effect on Si permeability. This study has thus led to the identification of residues that could play a key role in Si transport by the AQGPs and shown that substrate selectivity is likely ensured by more than one checkpoint within or near the pore.

Silicon (Si) is a beneficial substrate for many plants, conferring heightened resilience to environmental stress. A plant's ability to absorb Si is primarily dependent on the presence of a Si-permeable Lsi1 (NIP2-1) aquaporin in its roots. Structure-function analyses of Lsi1 channels from higher plants have thus far revealed two key molecular determinants of Si permeability: (a) the amino acid motif GSGR in the aromatic/arginine selectivity filter and (b) 108 amino acids between two highly conserved NPA domains. Curiously, tobacco (Nicotiana sylvestris) stands as a rare exception as it possesses an Lsi1 (NsLsi1) with these molecular signatures but is reported as a low Si accumulator. The aim of this study was therefore to identify whether additional determinants influence Si permeability via Lsi1 channels, focusing on the role of residues that differ uniquely in NsLsi1 relative to functional Lsi1 homologs. We observed tobacco indeed absorbed Si poorly (0.1% dw), despite NsLsi1 being expressed constitutively in planta. Si influx measured in NsLsi1-expressing Xenopus oocytes was very low (<13% that of OsLsi1 from rice (Oryza sativa) over a 3-hr time course), which likely explains why tobacco is a low Si accumulator. Interestingly, NsLsi1P125F displayed a significant gain of function (threefold increase in Si influx relative to NsLsi1WT), which coincided with a threefold increase in plasma membrane localization in planta, as measured by transient expression of GFP constructs in Nicotiana benthamiana leaves. These findings thus reveal a novel molecular determinant of Si transport in plants and inform breeding, biotechnological, and agricultural practices to effectively utilize Si in the context of plant resilience to environmental stress.

Atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G) have been linked to mutations in many of the proteins that are involved in alternative complement pathway activation. Age and etiology confounded, the prevalence of such mutations has been reported to be over 30 to 50% in these diseases. However, the cohorts studied included many children or individuals with a familial history of complement-related disorders and genetic tests were usually limited to exome sequencing of known causative or risk-associated genes. In this study, a retrospective adult cohort of 35 patients with biopsy-proven thrombotic microangiopathy (the largest in Canada) and 10 patients with C3 glomerulopathy was tested through an extended exome panel to identify causative defects in associated or candidate genes including those of the alternative and terminal complement pathways. A variant of unknown significance was also analyzed for pathogenicity through in vitro studies. To our surprise, the prevalence of known causative or risk-associated variants in either of these cohorts was found to be less than ~ 15% overall. However, the panel used and analyses carried out allowed to identify novel variants of potential clinical significance and a number of candidate genes. The prevalence of known genetic defects in adult-onset aHUS and C3G is thus probably much lower than 30 to 50%. Our results also point towards the importance of investigating diseases of the alternative complement pathway through extended exome panels and in vitro analyses. KEY MESSAGES: The alternative complement pathway plays a major role in the pathogenesis of hemolytic uremic syndrome and C3 glomerulopathy. Based on previous studies, both disorders have been commonly linked to variants in the various intermediates that sustain or regulate this pathway. The prevalence of such mutations in the adult-onset and sporadic forms of these diseases is probably much lower than expected based on larger series. The sporadic forms of complementopathies are likely to involve additional genes that are yet to be uncovered.

Bone turnover diseases are exceptionally prevalent in human and come with a high burden on physical health. While these diseases are associated with a variety of risk factors and causes, they are all characterized by common denominators, that is, abnormalities in the function or number of osteoblasts, osteoclasts, and/or osteocytes. As such, much effort has been deployed in the recent years to understand the signaling mechanisms of bone cell proliferation and differentiation with the objectives of exploiting the intermediates involved as therapeutic preys. Ion transport systems at the external and in the intracellular membranes of osteoblasts and osteoclasts also play an important role in bone turnover by coordinating the movement of Ca2+ , PO4 2- , and H+ ions in and out of the osseous matrix. Even if they sustain the terminal steps of osteoformation and osteoresorption, they have been the object of very little attention in the last several years. Members of the cation-Cl- cotransporter (CCC) family are among the systems at work as they are expressed in bone cells, are known to affect the activity of Ca2+ -, PO4 2- -, and H+ -dependent transport systems and have been linked to bone mass density variation in human. In this review, the roles played by the CCCs in bone remodeling will be discussed in light of recent developments and their potential relevance in the treatment of skeletal disorders.