Joost G. J. Hoenderop

TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption.

Mg2+ is an essential ion involved in a multitude of physiological and biochemical processes and a major constituent of bone tissue. Mg2+ homeostasis in mammals depends on the equilibrium between intestinal Mg2+ absorption and renal Mg2+ excretion, but little is known about the molecular nature of the proteins involved in the transepithelial transport of Mg2+ in these organs. Recently, it was shown that patients with mutations in TRPM6, a member of the transient receptor potential family of cation channels, suffer from hypomagnesemia with secondary hypocalcemia (HSH) as a result of impaired renal and/or intestinal Mg2+ handling. Here, we show that TRPM6 is specifically localized along the apical membrane of the renal distal convoluted tubule and the brush-border membrane of the small intestine, epithelia particularly associated with active Mg2+ (re)absorption. In kidney, parvalbumin and calbindin-D28K, two divalent-binding proteins, are co-expressed with TRPM6 and might function as intracellular Mg2+ buffers in the distal convoluted tubule. Heterologous expression of wild-type TRPM6 but not TRPM6 mutants identified in HSH patients induces a Mg2+- and Ca2+-permeable cation channel tightly regulated by intracellular Mg2+ levels. The TRPM6-induced channel displays strong outward rectification, has a 5-fold higher affinity for Mg2+ than for Ca2+, and is blocked in a voltage-dependent manner by ruthenium red. Our data indicate that TRPM6 comprises all or part of the apical Mg2+ channel of Mg2+-absorbing epithelia.

Permeation and Gating Properties of the Novel Epithelial Ca 2! Channel*

The recently cloned epithelial Ca2+ channel (ECaC) constitutes the Ca2+influx pathway in 1,25-dihydroxyvitamin D3-responsive epithelia. We have combined patch-clamp analysis and fura-2 fluorescence microscopy to functionally characterize ECaC heterologously expressed in HEK293 cells. The intracellular Ca2+ concentration in ECaC-expressing cells was closely correlated with the applied electrochemical Ca2+ gradient, demonstrating the distinctive Ca2+ permeability and constitutive activation of ECaC. Cells dialyzed with 10 mm1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid displayed large inward currents through ECaC in response to voltage ramps. The corresponding current-voltage relationship showed pronounced inward rectification. Currents evoked by voltage steps to potentials below −40 mV partially inactivated with a biexponential time course. This inactivation was less pronounced if Ba2+or Sr2+ replaced Ca2+ and was absent in Ca2+-free solutions. ECaC showed an anomalous mole fraction behavior. The permeability ratioP Ca:P Na calculated from the reversal potential at 30 mm[Ca2+] o was larger than 100. The divalent cation selectivity profile is Ca2+ > Mn2+ > Ba2+ ∼ Sr2+. Repetitive stimulation of ECaC-expressing cells induced a decay of the current response, which was greatly reduced if Ca2+ was replaced by Ba2+ and was virtually abolished if [Ca2+] o was lowered to 1 nm. In conclusion, ECaC is a Ca2+ selective channel, exhibiting Ca2+-dependent autoregulatory mechanisms, including fast inactivation and slow down-regulation.

CaT1 and the calcium release-activated calcium channel manifest distinct pore properties.

The calcium release-activated calcium channel (CRAC) is a highly Ca2+-selective ion channel that is activated on depletion of inositol triphosphate (IP3)-sensitive intracellular Ca2+ stores. It was recently reported that CaT1, a member of the TRP family of cation channels, exhibits the unique biophysical properties of CRAC, which led to the conclusion that CaT1 comprises all or part of the CRAC pore (Yue, L., Peng, J. B., Hediger, M. A., and Clapham, D. E. (2001) Nature 410, 705–709). Here, we directly compare endogenous CRAC with heterologously expressed CaT1 and show that they manifest several clearly distinct properties. CaT1 can be distinguished from CRAC in the following features: sensitivity to store-depleting agents; inward rectification in the absence of divalent cations; relative permeability to Na+ and Cs+; effect of 2-aminoethoxydiphenyl borate (2-APB). Moreover, CaT1 displays a mode of voltage-dependent gating that is fully absent in CRAC and originates from the voltage-dependent binding/unbinding of Mg2+ inside the channel pore. Our results imply that the pores of CaT1 and CRAC are not identical and indicate that CaT1 is a Mg2+-gated channel not directly related to CRAC.

80K-H as a New Ca2+ Sensor Regulating the Activity of the Epithelial Ca2+ Channel Transient Receptor Potential Cation Channel V5 (TRPV5)

The epithelial Ca2+ channel transient receptor potential cation channel V5 (TRPV5) constitutes the apical Ca2+ entry pathway in the process of active Ca2+ reabsorption. Ca2+ influx through TRPV5 is tightly controlled by modulators of Ca2+ homeostasis, including 1,25-dihydroxyvitamin D3 and dietary Ca2+. However, little is known about intracellular proteins that interact with TRPV5 and directly regulate the activation of this channel. By the use of cDNA microarrays, the present study identified 80K-H as the first protein involved in the Ca2+-dependent control of the epithelial Ca2+ channel TRPV5. 80K-H was initially identified as a protein kinase C substrate, but its biological function remains to be established. We demonstrated a specific interaction between 80K-H and TRPV5, co-localization of both proteins in the kidney, and similar transcriptional regulation by 1,25-dihydroxyvitamin D3 and dietary Ca2+. Furthermore, 80K-H directly bound Ca2+, and inactivation of its two EF-hand structures totally abolished Ca2+ binding. Electrophysiological studies using 80K-H mutants showed that three domains of 80K-H (the two EF-hand structures, the highly acidic glutamic stretch, and the His-Asp-Glu-Leu sequence) are critical determinants for TRPV5 activity. Importantly, inactivation of the EF-hand pair reduced the TRPV5-mediated Ca2+ current and increased the TRPV5 sensitivity to intracellular Ca2+, accelerating the feedback inhibition of the channel. None of the 80K-H mutants altered the TRPV5 plasma membrane localization nor the association of 80K-H with TRPV5, suggesting that 80K-H has a direct effect on TRPV5 activity. In conclusion, we report a novel function for 80K-H as a Ca2+ sensor controlling TRPV5 channel activity.

The Na+/Ca2+ Exchanger 1 (NCX1) Variant 3 as the Major Extrusion System in Renal Distal Tubular Transcellular Ca2+-Transport.

Background/Aims: Fine-tuning of renal calcium (Ca2+) reabsorption takes place in the late distal convoluted and connecting tubules (DCT2/CNT) of the kidney via transcellular Ca2+ transport. Here, Ca2+ enters the cell at the apical side via the epithelial Ca2+ channel transient receptor potential vanilloid 5 and is subsequently extruded at the basolateral side by the concerted actions of the plasma membrane Ca2+ ATPases and the Na+/Ca2+ exchanger 1 (NCX1). NCX1 is responsible for ∼70% of basolateral Ca2+ extrusion. The aim of this study was to determine the predominant NCX1 variant in the kidney and its role in Ca2+ transport. Methods: DCT2/CNT specific tubules were used to show the abundance of NCX1 specific isoforms. Renal NCX1 variants were cloned from mouse kidney tissue. Human Embryonic Kidney 293(T) cells were transiently transfected with NCX1.3, and Fura-2 measurements and 45Ca2+ uptake assays were performed to determine several characteristics of NCX1.3 in the reverse mode. Results: NCX1.3 was demonstrated to be the predominant NCX1 variant in the DCT2/CNT, next to NCX1.2 and NCX1.7. NCX1.3 could be inhibited by SN-6, an NCX-specific inhibitor, whereas stimulation of the cAMP/PKA or PKC-mediated pathway did not affect Ca2+ influx as measured in the reverse mode. Lowering intracellular Ca2+ concentrations resulted in a decreased Ca2+ uptake. Conclusion: NCX1.3 is the predominant NCX variant in the DCT2/CNT tubules. Its function is dependent on intracellular Ca2+ concentrations.