Voltage-Dependent Ca ²⁺ Channels

Voltage-dependent Ca ²⁺ channels have been well characterized in excitable tissues and at least four different types including, L, N, T and P, can be distinguished on the basis of electrophysiological and pharmacological properties. The molecular identity and precise localization of these Ca ²⁺ channels along the nephron is not well understood. Previous studies from Yu and colleagues have demonstrated the existence of voltage-dependent Ca ²⁺ channels in the kidney ( Table 62.1 ). They identified a gene transcript encoding the pore-forming alpha 1-subunit of a Ca ²⁺ channel (alpha 1A) in the glomerulus and the distal convoluted tubule (DCT). Skott and coworkers assessed the distribution of the identified alpha(1G)-subunit encoding a voltage-dependent Ca ²⁺ channel with T-type characteristics. RT-PCR analysis of microdissected rat nephron segments revealed alpha(1G) expression in DCT, CNT, cortical collecting duct (CCD), and inner medullary collecting duct (IMCD) ( Table 62.1 ). Immunohistochemistry on sections of rat kidneys using an anti-alpha(1G) antibody demonstrated labeling at the apical plasma membrane domains of DCT, CNT and IMCD principal cells. Another study from Bacskai and Friedman examined the parathyroid hormone (PTH)-sensitivity of Ca ²⁺ influx in single cultured cells originating from distal renal tubules by measuring the intracellular Ca ²⁺ concentration. They found that PTH activates dihydropyridine-sensitive Ca ²⁺ channels. Tan and Lau, described a 25 pS, cAMP-sensitive apical Ca ²⁺ channel in rabbit kidney CNT cells, that was inhibited by dihydropyridine agonists and depolarization. The properties of these Ca ²⁺ channels are largely conferred by their pore-forming alpha1 subunit. In general, voltage-sensitive Ca ²⁺ channels are heteromeric, multi-subunit proteins, as has been clearly demonstrated for the skeletal muscle and brain channels, and their properties are not only determined by the pore-forming alpha1-subunit itself, but also depend on the presence of regulatory beta subunits. To identify the accessory beta subunit of DCT/CNT Ca ²⁺ channels, degenerate primers based on published beta subunit sequences were used to amplify rat kidney cDNA by the polymerase chain reaction (PCR). Alternatively, spliced transcripts of three beta-subunit genes (beta-2,3,4) were identified. Northern blot analysis indicated that beta 4-subunit is preferentially expressed in kidney cortex. Transcripts of all three beta-subunit genes were detected by PCR in microdissected nephron segments, but only the beta 4-subunit was found in DCT/CNT. At present, the physiological significance of these voltage-dependent Ca ²⁺ channels in transepithelial Ca ²⁺ transport remains unclear. Functional Ca ²⁺ influx measurements were only performed in non-polarized single cell systems. In addition, pharmacological studies in polarized primary cultures of rabbit CNT and CCD have demonstrated that voltage-dependent Ca ²⁺ channel blockers do not inhibit transepithelial Ca ²⁺ transport. Additional studies are needed to address the functional relevance of voltage-dependent Ca ²⁺ channels in the process of transepithelial Ca ²⁺ transport in the kidney.

Other Ca ²⁺ Channels

Non-voltage-gated Ca ²⁺ channels in the kidney are mainly represented by the transient receptor potential (TRP) superfamily of proteins consisting of Ca ²⁺ permeable channels. TRP channels can be divided by sequence homology into at least six subfamilies, designated TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystins, PKD-type), TRPA (ankyrin), TRPML (mucolipin), and the more distant subfamily TRPN (no mechano-receptor potential or nomp). Mammalian homologues of the first identified Drosophila TRP gene encode a family of at least 20 ion channel proteins ( Figure 62.1 ). Mutations and/or dysfunction of TRP proteins produce many renal diseases, including Mg ²⁺ wasting, Ca ²⁺ -related disorders and polycystic kidney diseases.

Mammalian TRP family tree.

The evolutionary distance between the TRP channels is shown by the total branch lengths in point accepted mutations (PAM) units, which is the mean number of substitutions per 100 residues. The tree was calculated using the neighbor-joining method for human, rat and mouse sequences.

cDNA and Primary Protein Structure

The epithelial Ca ²⁺ channel family is restricted to two members and genomic cloning demonstrated that these Ca ²⁺ channels are transcribed from distinct genes juxtaposed on human chromosome 7q35, which suggests an evolutionary gene duplication event. TRPV5 and TRPV6 are completely conserved in terms of exon size in the coding regions and they share similar intron-exon structures with the other TRP genes. To date, epithelial Ca ²⁺ channels have been cloned from several species including pufferfish, rabbit, rat, mouse and human. TRPV5 and TRPV6 exhibit an overall homology of about 75%. Remarkably, several domains in TRPV5/6 are completely conserved within these species including the core structure of the protein consisting of 6 TM segments and the pore region, ankyrin repeats, PDZ motifs and putative protein kinase phosphorylation sites ( Figure 62.2 ). Four TRP-channel subunits will form the functional Ca ²⁺ channel. In addition, it was demonstrated that both epithelial channels are high mannose- and complex-glycosylated. PDZ motifs and ankyrin repeat domains are present in a diverse range of receptors and ion channels including other members of the TRPV subfamily. PDZ motifs are recognized by PDZ domains that are modular protein interaction domains playing a role in protein trafficking and protein complex assembly. In general, ankyrins connect ion channels, transporters and cell adhesion molecules to the spectrin-based cytoskeletal elements in distinct membrane domains.

The epithelial Ca ²⁺ channels are ~730 amino acids long with a predicted molecular mass of ~85 kDa. TRPV5 and TRPV6 contain a core domain consisting of 6 TM segments. In addition, a large cytosolic amino- and carboxyl-terminus are present containing ankyrin repeats. Between TM5 and TM6 there is a short hydrophobic stretch predicted to be the pore-forming region of these channels. Four channel subunits will form the functional Ca ²⁺ channel.

Distribution of TRPV5 and TRPV6

The renal expression profile of TRPV5 has been studied in detail. In different species, it was demonstrated that TRPV5 co-localizes with the Ca ²⁺ transport proteins, including calbindin-D _28K and the extrusion proteins, the plasma membrane ATPase 1b (PMCA1b) and the Na ⁺ -Ca ²⁺ exchanger type 1 (NCX1) in the late DCT (DCT2) and the CNT ( Figure 62.3 ). Transepithelial Ca ²⁺ transport is generally envisaged as a three step process consisting of passive entry of Ca ²⁺ across the apical membrane, cytosolic diffusion of Ca ²⁺ bound to calbindin-D _28K and active extrusion of Ca ²⁺ across the opposite basolateral membrane via PMCA1b and NCX1. The highest immunocytochemical abundance of TRPV5 was found in DCT2 with a gradual decrease along CNT. A minority of cells along CNT lacked immunopositive staining for TRPV5 and the other Ca ₂ ⁺ -transporting proteins. These negative cells were identified as intercalated cells.

Transepithelial Ca ²⁺ transport is carried out as a 3-step-process.

Following entry of Ca ²⁺ through the (hetero)tetrameric epithelial Ca ²⁺ channels, TRPV5 and TRPV6, Ca ²⁺ bound to calbindin diffuses to the basolateral membrane. At the basolateral membrane, Ca ²⁺ is extruded via a ATP-dependent Ca ²⁺ -ATPase (PMCA1b) and a Na ⁺ -Ca ²⁺ -exchanger (NCX1). In this way, there is net Ca ²⁺ transport from the luminal space to the extracellular compartment. The active form of vitamin D, 1,25-(OH) ₂ D ₃ , stimulates the individuals steps of transepithelial Ca ²⁺ transport by increasing the expression levels of the luminal Ca ²⁺ channels, calbindins and the extrusion systems.

In contrast to TRPV5, which is mainly expressed in the kidney, TRPV6 shows a more ubiquitous expression pattern. TRPV6 expression was predominantly found in the brush-border membrane of the small intestine where it co-localizes with calbindin-D _9K and PMCA1b in agreement with the expected properties being the gatekeeper in active Ca ²⁺ absorption. Recently, TRPV6 was detected in mouse kidney along the apical domain of DCT2 through inner medullary collecting duct (IMCD). TRPV6 co-localizes with TRPV5 and the other Ca ²⁺ transport proteins in DCT2 suggesting a role in Ca ²⁺ reabsorption. In addition, the protein is also detected in the intercalated cells and the IMCD that are not involved in transepithelial Ca ²⁺ transport pointing to additional functions of TRPV6. Thus, the precise role of this epithelial Ca ²⁺ channel in the kidney remains to be established, but given the widespread distribution of TRPV6 throughout the nephron segments functions could include Ca ²⁺ reabsorption, Ca ²⁺ signaling and others.

Biophysical Properties

In Human Embryonic Kidney (HEK) 293 cells heterogeneously expressing TRPV5, currents through the channel can be activated under conditions of high intracellular Ca ²⁺ buffering by hyperpolarizing voltage steps. In divalent cation free solutions, large inward currents are observed, which show a typical time-dependent increase (gating) that remains constant for more than one minute. Outward currents are negligible indicating that the channel is nearly completely inwardly rectifying. In analogy to voltage-dependent Ca ²⁺ channels, permeation through TRPV5/6 can by described by “repulsion” pore models considering a pore consisting of two high affinity binding sites, whereby the double occupation of the two binding sites by either Na ⁺ or Ca ²⁺ provides the “drive” for Ca ²⁺ conduction due to mutual repulsion of the two cations or by a three binding-site model in which two low affinity sites flank a high affinity binding site for Ca ²⁺ . Importantly, the current through TRPV5 and TRPV6 is carried exclusively by Ca ²⁺ at physiological extracellular Ca ²⁺ concentrations. In the situation where Ca ²⁺ is absent single channel Na ⁺ currents through TRPV5 can be measured in inside-out patches. Given the low conductance of these channels in the presence of extracellular Ca ²⁺ reliable single channel measurements are technically not feasible.

A characteristic aspect of currents through the epithelial Ca ²⁺ channels is the inactivation at negative membrane potentials. This inactivation is nearly complete if Ca ²⁺ is the charge carrier and is significantly delayed when Ca ₂ ⁺ is substituted for Ba ²⁺ or other divalents indicating that the process is Ca ²⁺ -dependent. Furthermore, currents of monovalent cations through these Ca ²⁺ channels do not inactivate. Repetitive stimulation of TRPV5 and TRPV6 currents by short hyperpolarizing pulses consequently result in a decay of the current. The slow recovery from inactivation in divalent cation free solution after the Ca ²⁺ -dependent decay is a further typical hallmark of these Ca ²⁺ channels. Although the above described structural aspects and basic electrophysiological properties of TRPV5 and TRPV6 are rather similar, some differences exist between these homologous channels including permeability of divalent cations, kinetics of Ca ²⁺ -dependent inactivation and recovery from inactivation. For instance, TRPV5 shows a higher permeability for Ba ²⁺ compared to TRPV6, e.g., the current ratio I _Ba /I _Ca is ~0.9 for TRPV5, but only ~0.4 for TRPV6. TRPV6 clearly shows a fast component of inactivation which is less prominent for TRPV5, e.g., time to 10% inactivation at hyperpolarizing steps to–100 mV is ~25 ms for TRPV5 but only ~40 ms for TRPV6. Interestingly, these typical differences could be explained by sequence differences between TRPV5 and TRPV6. Interestingly, the structural determinants of these differences appear not to be located in either the amino- or carboxyl-terminus, but in the intracellular TM2-TM3 linker sequence. Substitution of this TM2–TM3 linker of TRPV6 to TRPV5 confers the kinetic and permeation phenotype of TRPV6 to TRPV5, whereas exchanging the amino- or carboxyl-termini is ineffective. Mutation studies by Suzuki and co-workers demonstrated that a histidine residue at position 587 in the carboxyl-terminus close to TM segment 6 of TRPV6 affect the inactivation behavior of TRPV6. In addition some distinct pharmacological differences exist, e.g., ruthenium red is a 100-fold more potent blocker for TRPV5 than TRPV6 (IC ₅₀ ~9 μM for TRPV6 but~100 nM for TRPV5). Furthermore, TRPV5 is approximately four times more sensitive to Cd ²⁺ blockage than TRPV6 (IC ₅₀ ~70 nM for TRPV5 but~260 nM for TRPV6).

Mechanism of High Ca ²⁺ Selectivity

The high Ca ²⁺ selectivity of TRPV5 and TRPV6 refers to a mechanism of permeation as proposed previously for voltage-dependent Ca ²⁺ channels. In the absence of Ca ²⁺ , Mg ²⁺ will bind at a site within the pore-region, which is mainly determined by D542/D541. At low driving forces, e.g. less negative membrane potentials, the driving force is not sufficient to displace Mg ²⁺ out of the pore and the channel is blocked. At hyperpolarisation, Mg ²⁺ will be moved from this high affinity site thereby allowing monovalent cations to permeate the channels. Because of the higher affinity of the binding-site for Ca ²⁺ versus Mg ²⁺ , Ca ²⁺ will outnumber Mg ²⁺ at this site and is now blocking the pore for a movement of monovalent cations. If the inward driving force is sufficient enough to remove Ca ²⁺ from the high affinity site, a fast inward permeation occurs supported by the low affinity sites, which flank the high affinity Ca ²⁺ -binding site. All the data so far support the crucial role of D542/D541 in the channel pore region of TRPV5 and TRPV6 channels.

Rectification and Gating

A predominant inward rectification is a clear hallmark of the TRPV5 and TRPV6 channels besides the high Ca ²⁺ selectivity. This inward rectification is only partially due to blockage by Mg ²⁺ , which is removed by hyperpolarization. Thus, the most prominent part of rectification remains in the absence of intracellular Mg ²⁺ and is not due to blockage by endogenous polyamines. The intrinsic rectification of these channels is, however, dramatically reduced by neutralization of the negatively charged D542/D541 residues indicating that the permeation site is also involved in rectification. In mammalian cells heterogeneously expressing the epithelial Ca ²⁺ channels, TRPV5 and TRPV6 are constitutively open at a low intracellular Ca ²⁺ concentration and negative membrane voltage. The above described mechanism of removing channel blockage by Ca ²⁺ or Mg ²⁺ is necessarily part of the gating mechanism. At this moment other mechanisms that might influence gating of TRPV5 and TRPV6 cannot be excluded. Huang and colleagues showed that PIP ₂ activates TRPV5 and that activation of TRPV5 by PIP ₂ decreases the sensitivity of TRPV5 to inhibition by intracellular Mg ²⁺ .

Intracellular Ca ²⁺

TRPV5 and TRPV6 are controlled by a Ca ²⁺ -dependent feedback inhibition. Both Ca ²⁺ channels rapidly inactivate during hyperpolarizing voltage steps and this inhibition is reduced when Ca ²⁺ is substituted for Ba ²⁺ or Sr ²⁺ as charge carriers. The inactivation is dependent on the extracellular Ca ²⁺ concentration and occurred also in cells buffered intracellularly with BAPTA. In addition, the current disappeared during repetitive activation by short hyperpolarizing pulses. This current decay was significantly diminished when Ca ²⁺ was replaced by Ba ²⁺ as charge carrier and abolished when extracellular Ca ²⁺ was lowered to 1 nM, again indicating that a Ca ²⁺ -dependent process inhibits channel activity. Increase of the extracellular Ca ²⁺ concentration significantly stimulated the rate of current decay. Ca ²⁺ influx is a prerequisite for this phenomenon because the Ca ²⁺ -impermeable D542A mutant lacks a monovalent current decay in response to repetitive stimulation. These findings suggest that the TRPV5/6 channels are down-regulated by Ca ²⁺ influx through the channel and thus likely by increasing the Ca ²⁺ concentration in a micro-domain near the pore region, thereby inducing feedback inhibition of the channel. This could be a crucial mechanism for the regulation of TRPV5 and TRPV6 under physiological conditions. The inhibition is highly Ca ²⁺ -sensitive with calculated affinity values around 100 nM. Considering the high affinity mechanism of Ca ²⁺ -dependent TRPV5/6 inhibition, the presence of intracellular Ca ²⁺ buffering proteins, such as calbindins, will ultimately determine the channel activity.

Pharmacology

Ruthenium red and econazole are the most effective blockers of the epithelial Ca ²⁺ channels identified so far. The inorganic polycationic dye ruthenium red, which binds to phospholipids, inhibits TRPV5 and TRPV6 in a voltage-dependent manner. Transepithelial Ca ²⁺ transport in primary cultures of immunodissected rabbit CNT and CCD are inhibited in the same concentration range. Antimycotic imidazoles such as econazole and miconazole are highly effective inhibitors with IC ₅₀ values of the micromolar range. These compounds block the channels, however, voltage-independently. In addition, TRPV5 and TRPV6 are efficiently blocked by inorganic cations according to the following profile: Pb ²⁺ =Cu ²⁺ =Gd ³⁺ >Cd ²⁺ >Zn ²⁺ >La ³⁺ >Co ²⁺ >Fe ²⁺ >> Fe ³⁺ with IC ₅₀ values between 1 and 10 μM. Blockers of store-operated Ca ²⁺ entry channels (SOC), e.g., SKF965 and 2-aminoethoxydipheny borate (2-APB), are nearly ineffective for TRPV5 and TRPV6. Another SOC blocker, the adenylcyclase inhibitor MDL 12330A, exerts half-maximal inhibition of TRPV5 at ~20 μM. Xestospongin, a noncompetitive inositol (1,4,5)-trisphosphate receptor antagonist seems, however, to block TRPV6. Blockers of protein tyrosine kinases such as tyrphostin B46 and genistein, CaM antagonists calmidazolium R24571 and trifluoperazine, and voltage-dependent Ca ²⁺ channel blockers such as mibefradil, quinidine, nifedipine, niflumic acid and verapamil, have only small or no effects on TRPV5 activity. Interestingly, blockage by ruthenium red and Cd ²⁺ is much more sensitive for TRPV5 than TRPV6 which can, therefore, be used to differentiate both Ca ²⁺ channels.

pH

Previous studies indicated that acidification of the apical medium inhibits transepithelial Ca ²⁺ transport across primary cultures of rabbit CNT and CCD cells. ⁴⁵ Ca ²⁺ -uptake in TRPV5-expressing Xenopus laevis oocytes was inhibited by acidification of the incubation medium. Extracellular acidification reduced currents through TRPV5 carried by either monovalent or divalent cations, which was confirmed by Peng and colleagues Additionally, extracellular pH also affected current kinetics, extracellular Mg ²⁺ blockage and Ca ²⁺ affinity. The gating component of monovalent cation currents through TRPV5 was delayed at alkaline pH and as well as blockage by extracellular Mg ²⁺ . For voltage-dependent Ca ²⁺ channels and cyclic nucleotide-gated channels, it was found that protonation of a single glutamate residue in the pore region is responsible for the dramatic changes in divalent cation affinity by extracellular pH. A similar mechanism might be applicable for TRPV5 and TRPV6 in which a single aspartate residue in the pore determines the Ca ²⁺ permeation and Mg ²⁺ blockage of the channel. Extrapolating these findings to the in vivo situation, the pH influence could at least in part provide the molecular basis of acidosis-induced calciuresis. Interestingly, the molecular mechanism of TRPV5 blockage by protons is mechanistically understood taking into account the pore structure described above. Mutation of the glutamate residue at position 522 to glutamine preceding the pore helix decreased the inhibition of the channel by extracellular protons. Thus, pH sensitivity is mainly mediated by glutamate at position 522 and may act as the “pH sensor” of TRPV5.