Regulation of the epithelial calcium channel TRPV6 by the serum and glucocorticoid-inducible kinase isoforms SGK1 and SGK3

Epithelial calcium (re)absorption is mediated by TRPV5 and TRPV6 channels. TRPV5 is modulated by the SGK1 kinase, a process requiring the PDZ-domain containing scaffold protein NHERF2. The present study explored whether TRPV6 is similarly regulated by SGKs and the scaffold proteins NHERF1/2. In Xenopus oocytes, SGKs activate TRPV6 by increasing its plasma membrane abundance. Deletion of the putative PDZ binding motif on TRPV6 did not abolish channel activation by SGKs. Furthermore, coexpression of neither NHERF1 nor NHERF2 affected TRPV6 or potentiated the SGKs stimulating effect. The present observations disclose a novel TRPV6 regulatory mechanism which presumably participates in calcium homeostasis.     

Structural insights into the gating mechanisms of TRPV channels

Transient Receptor Potential channels from the vanilloid subfamily (TRPV) are a group of cation channels modulated by a variety of endogenous stimuli as well as a range of natural and synthetic compounds. Their roles in human health make them of keen interest, particularly from a pharmacological perspective. However, despite this interest, the complexity of these channels has made it difficult to obtain high resolution structures until recently. With the cryo-EM resolution revolution, TRPV channel structural biology has blossomed to produce dozens of structures, covering every TRPV family member and a variety of approaches to examining channel modulation. Here, we review all currently available TRPV structures and the mechanistic insights into gating that they reveal.     

TRPV5 and TRPV6 are expressed in placenta and bone tissues during pregnancy in mice

We investigated the dynamic expression of calcium transporters, TRPV5 and TRPV6, in placenta and bone to determine their role in maternal and fetal calcium balance during gestation. In placenta, TRPV5 was expressed predominantly in syncytiotrophoblasts of the labyrinthine zone, whereas TRPV6 was expressed in spongiotrophoblasts of the junction zone. In bone, the two transporters were found in osteoblasts, osteoclasts, cartilage and bone matrices. During the first half of gestation, TRPV5 and TRPV6 levels in bone were increased on pregnancy day (P) 0.5, then decreased on P3.5 followed by a slight increase on P6.5. During the second half of pregnancy, both the proteins and their mRNAs gradually increased from P9.5 to P15.5?P17.5 in both bone and placenta, followed at parturition by relatively high amounts in placenta, but markedly decreased amounts in bone. The expression pattern is likely related to the fetal and maternal calcium requirement during gestation, which may be regulated by estrogen and other hormones, because the fetal demand for calcium is greatest during the last few days of gestation for rats; maternal calcium metabolism is designed to meet the calcium needs of the fetus during this period. We found that TRPV5 and TRPV6 are involved in calcium transport in the placenta and bone, and therefore play a role in calcium homeostasis during embryonic and fetal development.     

Regulation of TRP channels by N-linked glycosylation

A subset of TRP channel proteins undergoes regulatory N-linked glycosylation. A glycosylation site in the first extracellular loop of TRPV5 is enzymatically cleaved by a secreted glucuronidase, indirectly regulating channel function. Members of the TRPC family share a similar site, although details about a regulatory role are lacking. A second conserved TRP channel glycosylation site is found immediately adjacent to the channel pore-forming loop; both TRPV1 and TRPV4--and perhaps other TRPV family members--are influenced by glycosylation at this site. N-linked glycosylation, and the dynamic regulation of this process, substantially impacts function and targeting of TRP channels.     

Basal cells express functional TRPV4 channels in the mouse nasal epithelium

Basal cells in the nasal epithelium (olfactory and airway epithelia) are stem/progenitor cells that are capable of dividing, renewing and differentiating into specialized cells. These stem cells can sense their biophysical microenvironment, but the underlying mechanism of this process remains unknown. Here, we demonstrate the prominent expression of the transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca²⁺-permeable channel that is known to act as a sensor for hypo-osmotic and mechanical stresses, in the basal cells of the mouse nasal epithelium. TRPV4 mRNA was expressed in the basal portions of the prenatal mouse nasal epithelium, and this expression continued into adult mice. The TRPV4 protein was also detected in the basal layers of the nasal epithelium in wild-type but not in TRPV4-knockout (TRPV4-KO) mice. The TRPV4-positive immunoreactions largely overlapped with those of keratin 14 (K14), a marker of basal cells, in the airway epithelium, and they partially overlapped with those of K14 in the olfactory epithelium. Ca²⁺ imaging analysis revealed that hypo-osmotic stimulation and 4α-phorbol 12,13 didecanoate (4α-PDD), both of which are TRPV4 agonists, caused an increase in the cytosolic Ca²⁺ concentration in a subset of primary epithelial cells cultured from the upper parts of the nasal epithelium of the wild-type mice. This response was barely noticeable in cells from similar parts of the epithelium in TRPV4-KO mice. Finally, there was no significant difference in BrdU-labeled proliferation between the olfactory epithelia of wild-type and TRPV4-KO mice under normal conditions. Thus, TRPV4 channels are functionally expressed in basal cells throughout the nasal epithelium and may act as sensors for the development and injury-induced regeneration of basal stem cells.     

Modulating TRPV4 channels with paclitaxel and lithium

Transient receptor potential V4 (TRPV4), a plasma membrane calcium channel, is implicated as a contributor to the initiation of chemotherapy-induced peripheral neuropathy (CIPN). Paclitaxel (PTX) is a commonly used anticancer drug that causes CIPN and lithium has been shown to prevent CIPN. However, the direct effect of PTX and lithium on TRPV4 is not clear. This study investigated these actions using biochemical, pharmacological, and electrophysiological approaches using a neuronal cell line (SH-SY5Y). The addition of pharmacologically appropriate levels of PTX increased the expression of TRPV4, TRPV4 currents, and TRPV4-dependent calcium fluxes. Prolonged exposure to PTX amplified the acute effects of TRPV4 expression, currents, and calcium fluxes. Pretreatment with lithium (1 mM) decreased TRPV4 currents and calcium fluxes in the absence and presence of PTX. These findings enhance our understanding of the properties and regulation of TRPV4, the cellular mechanisms of PTX-induced neuropathy, and the mechanism of lithium for prevention of CIPN.     

Modulation of TRPV4 by diverse mechanisms

Transient receptor potential ion channels (TRP) are a superfamily of non-selective ion channels which are opened in response to a diverse range of stimuli. The TRP vanilloid 4 (TRPV4) ion channel is opened in response to heat, mechanical stimuli, hypo-osmolarity and arachidonic acid metabolites. However, recently TRPV4 has been identified as an ion channel that is modulated by, and opened by intracellular signalling cascades from other receptors and signalling pathways. Although TRPV4 knockout mice show relatively mild phenotypes, some mutations in TRPV4 cause severe developmental abnormalities, such as the skeletal dyplasia and arthropathy. Regulated TRPV4 function is also essential for healthy cardiovascular system function as a potent agonist compromises endothelial cell function, leading to vascular collapse. A better understanding of the signalling mechanisms that modulate TRPV4 function is necessary to understand its physiological roles. Post translational modification of TRPV4 by kinases and other signalling molecules can modulate TRPV4 opening in response to stimuli such as mechanical and hyposmolarity and there is an emerging area of research implicating TRPV4 as a transducer of these signals as opposed to a direct sensor of the stimuli. Due to its wide expression profile, TRPV4 is implicated in multiple pathophysiological states. TRPV4 contributes to the sensation of pain due to hypo-osmotic stimuli and inflammatory mechanical hyperalsgesia, where TRPV4 sensitizaton by intracellular signalling leads to pain behaviors in mice. In the vasculature, TRPV4 is a regulator of vessel tone and is implicated in hypertension and diabetes due to endothelial dysfunction. TRPV4 is a key regulator of epithelial and endothelial barrier function and signalling to and opening of TRPV4 can disrupt these critical protective barriers. In respiratory function, TRPV4 is involved in cystic fibrosis, cilary beat frequency, bronchoconstriction, chronic obstructive pulmonary disease, pulmonary hypertension, acute lung injury, acute respiratory distress syndrome and cough.In this review we highlight how modulation of TRPV4 opening is a vital signalling component in a range of tissues and why understanding of TRPV4 regulation in the body may lead to novel therapeutic approaches to treating a range of disease states.     

TRPV5 and TRPV6 calcium channels in human T cells

The recent cloning of the special calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) has provided a molecular basis for studying previously unidentified calcium influx channels in electrically nonexcitable cells. In the present work using RT-PCR, we obtained the endogenous expression of mRNAs of genes trpv5 and trpv6 in lymphoblast leukemia Jurkat cells and in normal human T lymphocytes. Additionally, by immunoblotting, the presence of the channel-forming TRPV5 proteins has been shown both in the total lysate and in crude membrane fractions from Jurkat cells and normal T lymphocytes. The use of immunoprecipitation revealed TRPV6 proteins in Jurkat cells, whereas in normal T lymphocytes, this protein was not detected. The expression pattern and the selective Ca²⁺ permeation properties of TRPV5 and TRPV6 channels indicate the important role of these channels in Ca²⁺ homeostasis, as well as most likely in malignant transformation of blood cells.     

Discovery and characterization of a positive allosteric modulator of transient receptor potential canonical 6 (TRPC6) channels

The non-selective second messenger-gated cation channel TRPC6 (transient receptor potential canonical 6) is activated by diacylglycerols (DAG) in a PKC-independent manner and plays important roles in a variety of physiological processes and diseases. In order to facilitate novel therapies, the development of potent inhibitors as well as channel-activating agents is of great interest. The screening of a chemical library, comprising about 17,000 small molecule compounds, revealed an agent, which induced increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in a concentration-dependent manner (EC₅₀ = 2.37 ± 0.25 μM) in stably TRPC6-expressing HEK293 cells. This new compound (C20) selectively acts on TRPC6, unlike OAG (1-oleoyl-1-acetyl-sn-glycerol), which also activates PKC and does not discriminate between TRPC6 and the closely related channels TRPC3 and TRPC7. Further evaluation by Ca²⁺ assays and electrophysiological studies revealed that C20 rather operated as an enhancer of channel activation than as an activator by itself and led to the assumption that the compound C20 is an allosteric modulator of TRPC6, enabling low basal concentrations of DAG to induce activation of the ion channel. Furthermore, C20 was tested in human platelets that express TRPC6. A combined activation of TRPC6 with C20 and OAG elicited a robust increase in [Ca²⁺]_i in human platelets. This potentiated channel activation was sensitive to TRPC6 channel blockers. To achieve sufficient amounts of C20 for biological studies, we applied a one-pot synthesis strategy. With regard to studies in native systems, the sensitizing ability of C20 can be a valuable pharmacological tool to selectively exaggerate TRPC6-dependent signals.     

Wasted TMEM16A channels are rescued by phosphatidylinositol 4,5-bisphosphate

Recently there has been a flurry of interest in the regulation of the homo-dimeric calcium-activated chloride channel ANO1 (also known as TMEM16A) by phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P₂). These recent studies show that upon Ca²⁺ binding, PI(4,5)P₂ cooperates to maintain the conductive state of ANO1. PI(4,5)P₂ does so by binding to sites or modules on the protein’s cytosolic side. These findings add a new function to the PI(4,5)P₂ repertoire and a new dimension to ANO1 gating.     

The gating of single calcium-dependent potassium channels is described by an activation/blockade mechanism

Single calcium dependent potassium channels from cultured rat myoballs have been studied with the patch clamp technique, and current records subjected to statistical analysis. From the dependence of the mean open state probability on the internal calcium concentration, two calcium ions are required to open the channel. The open state and closed state lifetime distributions reveal that the usual activation model is not applicable to these channels. They are consistent with a two step gating mechanism that involves both activation by calcium and blockade by a calcium-sensitive gate.     

Mg2+-dependent gating and strong inward rectification of the cation channel TRPV6

TRPV6 (CaT1/ECaC2), a highly Ca(2+)-selective member of the TRP superfamily of cation channels, becomes permeable to monovalent cations in the absence of extracellular divalent cations. The monovalent currents display characteristic voltage-dependent gating and almost absolute inward rectification. Here, we show that these two features are dependent on the voltage-dependent block/unblock of the channel by intracellular Mg(2+). Mg(2+) blocks the channel by binding to a site within the transmembrane electrical field where it interacts with permeant cations. The block is relieved at positive potentials, indicating that under these conditions Mg(2+) is able to permeate the selectivity filter of the channel. Although sizeable outward monovalent currents were recorded in the absence of intracellular Mg(2+), outward conductance is still approximately 10 times lower than inward conductance under symmetric, divalent-free ionic conditions. This Mg(2+)-independent rectification was preserved in inside-out patches and not altered by high intracellular concentrations of spermine, indicating that TRPV6 displays intrinsic rectification. Neutralization of a single aspartate residue within the putative pore loop abolished the Mg(2+) sensitivity of the channel, yielding voltage-independent, moderately inwardly rectifying monovalent currents in the presence of intracellular Mg(2+). The effects of intracellular Mg(2+) on TRPV6 are partially reminiscent of the gating mechanism of inwardly rectifying K(+) channels and may represent a novel regulatory mechanism for TRPV6 function in vivo.     

Human TRPV5 and TRPV6: Key players in cadmium and zinc toxicity

TRPV5 and TRPV6 are two major calcium transport pathways in the human body maintaining calcium homeostasis. TRPV5 is mainly expressed in the distal convoluted and connecting tubule where it is the major, regulated pathway for calcium reabsorption. TRPV6 serves as an important calcium entry pathway in the duodenum and the placenta. Previously, we showed that human TRPV6 (hTRPV6) transports several heavy metals. In this study we tested whether human TRPV5 (hTRPV5) also transports cadmium and zinc, and whether hTRPV5 together with hTRPV6 are involved in cadmium and zinc toxicity. The hTRPV5 mRNA and protein were expressed in HEK293 cells transiently transfected with pTagRFP-C1-hTRPV5. The overexpression of the hTRPV5 protein at the plasma membrane was revealed by cell surface biotinylation and immunofluorescence techniques. We observed that both cadmium and zinc permeate hTRPV5 in ion imaging experiments using Fura-2 or Newport Green DCF. Our results were further confirmed using whole-cell patch clamp technique. Transient overexpression of hTRPV5 or hTRPV6 sensitized cells to cadmium and zinc. Toxicity curves of cadmium and zinc were also shifted in hTRPV6 expressing HEK293 cells clones. Our results suggest that TRPV5 and TRPV6 are crucial gates controlling cadmium and zinc levels in the human body especially under low calcium dietary conditions, when these channels are maximally upregulated.     

A molecular mechanism for proton-dependent gating in KcsA

Activation gating in KcsA is elicited by changes in intracellular proton concentration. Thompson et al. [1] identified a charge cluster around the inner gate that plays a key role in defining proton activation in KcsA. Here, through functional and spectroscopic approaches, we confirmed the role of this charge cluster and now provide a mechanism of pH-dependent gating. Channel opening is driven by a set of electrostatic interactions that include R117, E120 and E118 at the bottom of TM2 and H25 at the end of TM1. We propose that electrostatic compensation in this charge cluster stabilizes the closed conformation at neutral pH and that its disruption at low pH facilitates the transition to the open conformation by means of helix-helix repulsion.     

Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+).

Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.     

Role of the Conserved Lysine Residue in the Middle of the Predicted Extracellular Loop Between M2 and M3 in the GABAA Receptor

Abstract : In α1, β2, and γ2 subunits of the γ-aminobutyric acid A (GABA_A) receptor, a conserved lysine residue occupies the position in the middle of the predicted extracellular loop between the transmembrane M2 and M3 regions. In all three subunits, this residue was mutated to alanine. Whereas the mutation in α1 and β2 subunits results each in about a sixfold shift of the concentration-response curve for GABA to higher concentrations, no significant effect by mutation in the γ subunit was detected. The affinity for the competitive inhibitor bicuculline methiodide was not affected by the mutations in either the α1 subunit or the β2 subunit. Concentration-response curves for channel activation by pentobarbital were also shifted to higher concentrations by the mutation in the α and β subunits. Binding of [³H]Ro 15-1788 was unaffected by the mutation in the α subunit, whereas the binding of [³H]muscimol was shifted to lower affinity. Mutation of the residue in the α1 subunit to E, Q, or R resulted in an about eight-, 10-, or fivefold shift, respectively, to higher concentrations of the concentration-response curve for GABA. From these observations, it is concluded that the corresponding residues on the α1 and β2 subunits are involved more likely in the gating of the channel by GABA than in the binding of GABA or benzodiazepines.     

Cooperativeness of Orai cytosolic domains tunes subtype-specific gating

Activation of immune cells is triggered by the Ca(2+) release-activated Ca(2+) current, which is mediated via channels of the Orai protein family. A key gating process of the three Orai channel isoforms to prevent Ca(2+) overload is fast inactivation, most pronounced in Orai3. A subsequent reactivation is a unique gating characteristic of Orai1 channels, whereas Orai2 and Orai3 currents display a second, slow inactivation phase. Employing a chimeric approach by sequential swapping of respective intra- and extracellular regions between Orai1 and Orai3, we show here that Orai1 specific proline/arginine-rich domains in the N terminus mediate reactivation, whereas the second, intracellular loop modulates fast and slow gating processes. Swapping C-terminal strands lacks a significant impact. However, simultaneous transfer of Orai3 N terminus and its second loop or C terminus in an Orai1 chimera substantially increases fast inactivation centered between wild-type channels. Concomitant swap of all three cytosolic strands from Orai3 onto Orai1 fully conveys Orai3-like gating characteristics, in a strongly cooperative manner. In conclusion, Orai subtype-specific gating requires a cooperative interplay of all three cytosolic domains.     

Autocrine activation of ion currents in the two-cell mouse embryo

The actions of autocrine ligands are required for the normal development of the preimplantation embryo in vitro. These ligands act as survival factors for the preimplantation stage embryo. One autocrine ligand, paf (1-o-alkyl-2-acetyl-sn-gylcero-3-phosphocholine), induced a dihydropyridine-sensitive calcium transient in the zygote and two-cell embryo, and these transients were required for the normal preimplantation stage survival. Paf induces an influx of external calcium through a dihydropyridine-sensitive channel. Dihydropyridine-sensitive currents are voltage-regulated, yet to date there is no evidence of membrane voltage depolarization in the two-cell embryo. To define the paf-induced calcium influx we have examined the response of the membrane potential and ion currents to paf in two-cell embryos. An initial response to paf challenge was the expression of an ion current (-15.6+/-1.6 pA) that was dependent upon extracellular calcium, was not voltage-gated but was dihydropyridine (nifedipine)-sensitive. This calcium current was followed (91+/-6 s after paf) by a net outward current (284+/-59 pA) that was composed of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate-sensitive (anion channel blocker) and tetraethylammonium chloride-sensitive (K(+) channel blocker) currents. This current corresponded temporally with a marked paf-induced transient hyperpolarization of the membrane potential (-8.4+/-1.2 mV) that was dependent upon the generation of the calcium transient. The results directly demonstrate the activation of a voltage-independent calcium current in response to paf and show for the first time the expression of an afterhyperpolarization that occurs as a response to the calcium transient.