René J. M. Bindels

Introduction and Aims: Magnesium (Mg2+) is an essential mineral for cell growth, neuroplasticity and muscle contraction. Blood Mg2+ levels below 0.7 mmol/L may cause a heterogeneous clinical phenotype, including muscle cramps, epilepsy and disturbances in K+ and Ca2+ homeostasis. Over the last decade, the genetic origin of several familial forms of hypomagnesemia has been delineated. In 2000, mutations in FXYD2, encoding the γ-subunit of the Na+-K+-ATPase, were identified to cause isolated dominant hypomagnesemia (IDH) in a large Dutch family suffering from hypomagnesemia, hypocalciuria and chondrocalcinosis. However, additional patients were never identified.

The kidney plays an important role in maintaining the systemic Ca2+ and Mg2+ balance. Thus the renal reabsorptive capacity of these cations can be amended to adapt to disturbances in plasma Ca2+ and Mg2+ concentrations. The reabsorption of Ca2+ and Mg2+ is driven by transport of other electrolytes, sometimes through selective channels and often supported by hormonal stimuli. It is, therefore, not surprising that monogenic disorders affecting such renal processes may impose a shift in, or even completely blunt, the reabsorptive capacity of these divalent cations within the kidney. Accordingly, in Dent's disease, a disorder with defective proximal tubular transport, hypercalciuria is frequently observed. Dysfunctional thick ascending limb transport in Bartter's syndrome, familial hypomagnesaemia with hypercalciuria and nephrocalcinosis, and diseases associated with Ca2+-sensing receptor defects, markedly change tubular transport of Ca2+ and Mg2+. In the distal convolutions, several proteins involved in Mg2+ transport have been identified [TRPM6 (transient receptor potential melastatin 6), proEGF (pro-epidermal growth factor) and FXYD2 (Na+/K+-ATPase γ-subunit)]. In addition, conditions such as Gitelman's syndrome, distal renal tubular acidosis and pseudohypoaldosteronism type II, as well as a mitochondrial defect associated with hypomagnesaemia, all change the renal handling of divalent cations. These hereditary disorders have, in many cases, substantially increased our understanding of the complex transport processes in the kidney and their contribution to the regulation of overall Ca2+ and Mg2+ balance.

Primary hypomagnesemia is a heterogeneous group of disorders characterized by renal or intestinal magnesium (Mg²⁺) wasting, resulting in tetany, cardiac arrhythmias, and seizures. The kidney plays an essential role in maintaining blood Mg²⁺ levels, with a prominent function for the Mg²⁺-transporting channel transient receptor potential cation channel, subfamily M, member 6 (TRPM6) in the distal convoluted tubule (DCT). In the DCT, Mg²⁺ reabsorption is an active transport process primarily driven by the negative potential across the luminal membrane. Here, we studied a family with isolated autosomal dominant hypomagnesemia and used a positional cloning approach to identify an N255D mutation in KCNA1, a gene encoding the voltage-gated potassium (K⁺) channel Kv1.1. Kv1.1 was found to be expressed in the kidney, where it colocalized with TRPM6 along the luminal membrane of the DCT. Upon overexpression in a human kidney cell line, patch clamp analysis revealed that the KCNA1 N255D mutation resulted in a nonfunctional channel, with a dominant negative effect on wild-type Kv1.1 channel function. These data suggest that Kv1.1 is a renal K⁺ channel that establishes a favorable luminal membrane potential in DCT cells to control TRPM6-mediated Mg²⁺ reabsorption.

Recent identification of a mutation in the EGF gene that causes isolated recessive hypomagnesemia led to the finding that EGF increases the activity of the epithelial magnesium (Mg²⁺) channel transient receptor potential M6 (TRPM6). To investigate the molecular mechanism mediating this effect, we performed whole-cell patch-clamp recordings of TRPM6 expressed in human embryonic kidney 293 (HEK293) cells. Stimulation of the EGF receptor increased current through TRPM6 but not TRPM7. The carboxy-terminal α-kinase domain of TRPM6 did not participate in the EGF receptor–mediated increase in channel activity. This activation relied on both the Src family of tyrosine kinases and the downstream effector Rac1. Activation of Rac1 increased the mobility of TRPM6, assessed by fluorescence recovery after photobleaching, and a constitutively active mutant of Rac1 mimicked the stimulatory effect of EGF on TRPM6 mobility and activity. Ultimately, TRPM6 activation resulted from increased cell surface abundance. In contrast, dominant negative Rac1 decreased TRPM6 mobility, abrogated current development, and prevented the EGF-mediated increase in channel activity. In summary, EGF-mediated stimulation of TRPM6 occurs via signaling through Src kinases and Rac1, thereby redistributing endomembrane TRPM6 to the plasma membrane. These results describe a regulatory mechanism for transepithelial Mg²⁺ transport and consequently whole-body Mg²⁺ homeostasis.

A recently described novel controllable method to regulate protein expression is based on a mutated FK506-binding protein-12 (mtFKBP) that is unstable and rapidly degraded in mammalian cells. This instability can be conferred to other proteins directly fused to mtFKBP. Binding of a synthetic cell-permeant ligand (Shield-1) to mtFKBP reverses the instability, allowing conditional expression of mtFKBP-fused proteins. We adapted this strategy to study multimeric plasma membrane proteins using the ion channel TRPV5 as model protein. mtFKBP-TRPV5 forms functional ion channels and its expression can be controlled in a time- and dose-dependent fashion using Shield-1. Moreover, in the presence of Shield-1, mtFKBP-TRPV5 formed heteromultimeric channels with untagged TRPV5, which were codegraded upon washout of Shield-1, providing a strategy to study multimeric plasma membrane protein complexes without the need to destabilize all individual subunits.

TRPV5 is a Ca(2+)-selective channel involved in transcellular Ca(2+) absorption expressed in kidney and in the ruffled border of osteoclasts. Studies in hypercalciuric TRPV5 knockout (TRPV5(-/-)) mice, which display significantly increased vitamin D levels, showed that TRPV5 ablation increases number and size of osteoclasts but impairs osteoclast-mediated bone resorption. The latter is not in line with the observed decreased bone thickness in TRPV5(-/-) mice. Bisphosphonates also inhibit osteoclast-mediated bone resorption. The aim of this study was to evaluate the effect of alendronate on the expression of the Ca(2+) transporters in bone, kidney, and duodenum and, importantly, the bone phenotype in TRPV5(-/-) mice. Wildtype (TRPV5(+/+)) and TRPV5(-/-) mice were treated during 10 wk with 2 mg/kg alendronate or vehicle weekly and housed in metabolic cages at the end of treatment. Urine and blood samples were taken for biochemical analysis, and duodenum, kidney, and femur were sampled. Expression of Ca(2+) transporters and osteoclast ruffled border transporters in bone and cultured osteoclasts was determined by QPCR analysis. Femurs were scanned using muCT, and resorption pit assays were performed in bone marrow cultures isolated from TRPV5(+/+) and TRPV5(-/-) mice. Alendronate treatment enhanced bone thickness in TRPV5(+/+) mice but also normalized the disturbed bone morphometry parameters in TRPV5(-/-) mice. Bone TRPV5 expression was specifically enhanced by alendronate, whereas the expression of Ca(2+) transporters in kidney and intestine was not altered. The expression of the osteoclast ruffled border membrane proteins chloride channel 7 (CLC-7) and the vacuolar H(+)-ATPase did not differ between both genotypes, but alendronate significantly enhanced the expression and PTH levels in TRPV5(-/-) mice. The expression of TRPV5, CLC-7, and H(+)-ATPase in osteoclast cultures was not affected by alendronate. The number of resorption pits was reduced in TRPV5(-/-) bone marrow cultures, but the response to vitamin D was similar to that in TRPV5(+/+) cultures. The alendronate-induced upregulation of TRPV5 in bone together with the decreased resorptive capacity of TRPV5(-/-) osteoclasts in vitro suggests that TRPV5 has an important role in osteoclast function. However, our data indicate that significant bone resorption still occurs in TRPV5(-/-) mice, because alendronate treatment normalized bone thickness in these mice. Thus, TRPV5(-/-) mice are able to rescue the resulting defect in osteoclast-mediated bone resorption, possibly mediated by the long-term hypervitaminosis D or other (non)hormonal compensatory mechanisms.

The distal convoluted tubule (DCT) is the shortest segment of the nephron and consists of an early (DCT1) and late part (DCT2). Here, several transport proteins, like the thiazide-sensitive NaCl cotransporter (NCC) and the epithelial magnesium (Mg²⁺) channel (TRPM6), are exclusively expressed. This makes the DCT the major site of active transcellular Mg²⁺ reabsorption determining the final excretion in the urine. Following the Mg²⁺ influx via the apically localized TRPM6, intracellular Mg²⁺ diffuses to the basolateral membrane where it is extruded to the blood compartment via still-unidentified Mg²⁺ transporters. Recent years have witnessed multiple breakthroughs in the field of transcellular Mg²⁺ reabsorption. Epidermal growth factor and estrogen were identified as magnesiotropic hormones by their effect on TRPM6 activity. Intracellularly, receptor of activated protein kinase C 1 and adenosine triphosphate were shown to inhibit TRPM6 activity through its α-kinase domain. Furthermore, dysregulation or malfunction of transcellular Mg²⁺ reabsorption in DCT has been associated with renal Mg²⁺ wasting. Mutations in TRPM6 are responsible for hypomagnesemia with secondary hypocalcemia. A defect in the γ-subunit of the Na⁺/K⁺-adenosine triphosphatase causes isolated dominant hypomagnesemia resulting from renal Mg²⁺ wasting. Moreover, in Gitelman’s syndrome, mutations in NCC also result in impaired transcellular Mg²⁺ reabsorption in DCT. This review highlights our recently obtained knowledge concerning the molecular regulation of transcellular Mg²⁺ reabsorption.