Molecular mechanisms, physiological roles, and therapeutic implications of ion fluxes in bone cells: Emphasis on the cation-Cl- cotransporters.

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.

Anatomophysiology of the Henle's Loop: Emphasis on the Thick Ascending Limb.

The loop of Henle plays a variety of important physiological roles through the concerted actions of ion transport systems in both its apical and basolateral membranes. It is involved most notably in extracellular fluid volume and blood pressure regulation as well as Ca2+ , Mg2+ , and acid-base homeostasis because of its ability to reclaim a large fraction of the ultrafiltered solute load. This nephron segment is also involved in urinary concentration by energizing several of the steps that are required to generate a gradient of increasing osmolality from cortex to medulla. Another important role of the loop of Henle is to sustain a process known as tubuloglomerular feedback through the presence of specialized renal tubular cells that lie next to the juxtaglomerular arterioles. This article aims at describing these physiological roles and at discussing a number of the molecular mechanisms involved. It will also report on novel findings and uncertainties regarding the realization of certain processes and on the pathophysiological consequences of perturbed salt handling by the thick ascending limb of the loop of Henle. Since its discovery 150 years ago, the loop of Henle has remained in the spotlight and is now generating further interest because of its role in the renal-sparing effect of SGLT2 inhibitors. © 2022 American Physiological Society. Compr Physiol 12:1-21, 2022.

Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters.

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.

Molecular characteristics and physiological roles of Na+ -K+ -Cl- cotransporter 2.

Na+ -K+ -Cl- cotransporter 2 (NKCC2; SLC12A1) is an integral membrane protein that comes as three splice variants and mediates the cotranslocation of Na+ , K+ , and Cl- ions through the apical membrane of the thick ascending loop of Henle (TALH). In doing so, and through the involvement of other ion transport systems, it allows this nephron segment to reclaim a large fraction of the ultrafiltered Na+ , Cl- , Ca2+ , Mg2+ , and HCO3- loads. The functional relevance of NKCC2 in human is illustrated by the many abnormalities that result from the inactivation of this transport system through the use of loop diuretics or in the setting of inherited disorders. The following presentation aims at discussing the physiological roles and molecular characteristics of Na+ -K+ -Cl- cotransport in the TALH and those of the individual NKCC2 splice variants more specifically. Many of the historical and recent data that have emerged from the experiments conducted will be outlined and their larger meaning will also be placed into perspective with the aid of various hypotheses.

Endocytic recycling of Na+ -K+ -Cl- cotransporter type 2: importance of exon 4.

KEY POINTS: Na+ -K+ -Cl- cotransporter type 2 (NKCC2) is a 27-exon membrane protein that is expressed in the thick ascending limb (TAL) of Henle where it is involved in reabsorption of the ultrafiltered NaCl load. It comes as three splice variants that are identical to each other except for the residue composition of exon 4 and that differ in their transport characteristics, functional roles and distributions along the TAL. In this report, it is shown that the variants also differ in their trafficking properties and that two residues in exon 4 play a key role in this regard. One of these residues was also shown to sustain carrier internalization. Through these results, a novel function for the alternatively spliced exon of NKCC2 has been identified and a domain that is involved in carrier trafficking has been uncovered for the first time in a cation-Cl- cotransporter family member. ABSTRACT: Na+ -K+ -Cl- cotransporter type 2 (NKCC2) is a 12-transmembrane (TM) domain cell surface glycoprotein that is expressed in the thick ascending limb (TAL) of Henle and stimulated during cell shrinkage. It comes as three splice variants (A, B and F) that are identical to each other except for TM2 and the following connecting segment (CS2). Yet, these variants do not share the same localization, transport characteristics and physiological roles along the TAL. We have recently found that while cell shrinkage could exert its activating effect by increasing NKCC2 expression at the cell surface, the variants also responded differentially to this stimulus. In the current work, a mutagenic approach was exploited to determine whether CS2 could play a role in carrier trafficking and identify the residues potentially involved. We found that when the residue of position 238 in NKCC2A (F) and NKCC2B (Y) was replaced by the corresponding residue in NKCC2F (V), carrier activity increased by over 3-fold and endocytosis decreased concomitantly. We also found that when the residue of position 230 in NKCC2F (M) was replaced by the one in NKCC2B (T), carrier activity and affinity for ions both increased substantially whereas expression at the membrane decreased. Taken together, these results suggest that CS2 is involved in carrier trafficking and that two of its residues, those of positions 238 and 230, are part of an internalization motif. They also indicate that the divergent residue of position 230 plays the dual role of specifying ion affinity and sustaining carrier internalization.

Regulation of Na+-K+-Cl- cotransporter type 2 by the with no lysine kinase-dependent signaling pathway.

Na+-K+-Cl- cotransporter type 2 (NKCC2) is confined to the apical membrane of the thick ascending limb of Henle, where it reabsorbs a substantial fraction of the ultrafiltered NaCl load. It is expressed along this nephron segment as three main splice variants (called NKCC2A, NKCC2B, and NKCC2F) that differ in residue composition along their second transmembrane domain and first intracellular cytosolic connecting segment (CS2). NKCC2 is known to be activated by cell shrinkage and intracellular [Cl-] reduction. Although the with no lysine (WNK) kinases could play a role in this response, the mechanisms involved are ill defined, and the possibility of variant-specific responses has not been tested thus far. In this study, we have used the Xenopus laevis oocyte expression system to gain further insight in these regards. We have found for the first time that cell shrinkage could stimulate NKCC2A- and NKCC2B-mediated ion transport by increasing carrier abundance at the cell surface and that this response was achieved (at least in part) by the enzymatic function of a WNK kinase. Interestingly, we have also found that the activity and cell surface abundance of NKCC2F were less affected by cell shrinkage compared with the other variants and that ion transport by certain variants could be stimulated through WNK kinase expression in the absence of carrier redistribution. Taken together, these results suggest that the WNK kinase-dependent pathway can affect both the trafficking as well as intrinsic activity of NKCC2 and that CS2 plays an important role in carrier regulation.