REGULATION OF THE HUMAN SHORT TRANSIENT RECEPTOR POTENTIAL CHANNEL 3 (hTRP3) BY THE SERUM- AND GLUCOCORTICOID-INDUCED KINASE 1 (SGK1)

Assiut University web-site: www.aun.edu.eg

REGULATION OF THE HUMAN SHORT TRANSIENT RECEPTOR POTENTIAL CHANNEL 3 (hTRP3) BY THE SERUM- AND GLUCOCORTICOID-INDUCED

KINASE 1 (SGK1)

HAMDY M. EMBARK

Animal Physiology Department, Faculty of Vet. Medicine, South Valley University, Qena, Egypt

Received: 4 July 2016;       Accepted:  9 August 2016

INTRODUCTION

Calcium ions (Ca²⁺) are essential for many physiological processes, including fertilization, muscle contraction, hormone secretion, immune responses, brain functions, cell growth and last but not least, apoptosis(Orreniuset al., 2003; Zhang et al., 2015). The signal transduction capacity of Ca²⁺ depends on the 10,000-fold gradient across the plasma membrane with 2.5 mM extracellular and resting intracellular Ca²⁺ ion concentration of approximately 100 nM (Clapham, 1995). Increased cytosolic free Ca²⁺levels are interposed by either Ca²⁺ inflow across the plasma membrane by using different Ca²⁺ channels (Barrittet al., 2008) orrelease of Ca²⁺ from the internal Ca²⁺stores such as the endoplasmic Reticulum (ER) (Parekh and Penner, 1997; Verkhratsky and Petersen, 2002).

Corresponding author:Dr.HAMDY M.EMBARK

E-mail address:h.embark@vet.svu.edu.eg

Present address: Animal Physiology Department, Faculty of Vet. Medicine, South Valley University, Qena, Egypt

In mammals, Ca²⁺ concentration gradient between intracellular and extracellular fluids is tightly regulated by a complex homeostatic control mechanism involving fluxes of Ca²⁺ between theinterstitialfluid and the intestine, kidney, and bone (Mundy and Guise, 1999).The regulation of these fluxes islikely to come from more careful controlof three important hormones: thyrocalcitonin, parathyroid hormone (PTH), and Calcitriol (Boden and Kaplan, 1990). Several important cellular functions are dependent on the maintenance of the extracellular Ca²⁺ concentration within a very narrow range (Brown and MacLeod, 2001). Importantly, disturbances of this tightly controlled homeostatic system causesupsets inthe body's calcium metabolism that have expected effects, which can be attributed to effects on these cellular functions (Peacock, 2010).

Activation of the body's cells by hormones, neurotransmitters, and agents that stimulate the activity of the enzyme phospholipase C (PLC) leads to release of Ca²⁺ from distinct intracellular Ca²⁺ stores (Fan and Byron, 2000) and is followed by passive influx of Ca²⁺ from the extracellular space via a group of channels that have differently been referred to as Ca²⁺ release activated Ca²⁺ channels (CRAC), store operated Ca²⁺ channels (SOC), and receptor operated Ca²⁺ channels (ROC) (Hoth and Penner, 1992; Zweifach and Lewis, 1995).

These store-operated channels serve refilling of the Ca²⁺ store by providing a Ca²⁺ entry pathway and may in addition control cell membrane potential and homeostasis of monovalent cations (Parekh and Penner, 1997). Because Ca²⁺ entering the cell replenishes the intracellular Ca²⁺ stores that act likes capacitors, it has also been called “CapacitativeCa²⁺ Entry, (CCE)” or “Store-Operated Ca²⁺ Entry, (SOCE)” (Putney, 1990).Transient Receptor Potential (TRP) ion channels or TRPs are thought to mediate CCE or SOCE (Montell, 1997).

TRPsare the largest group of non-selective and polymodalcation channels which pass Ca²⁺ (and other cations too) into the cell down its electrochemical gradient in response to different stimuli, thereby increasing the intracellular Ca²⁺ concentration and causing cell depolarization (Kumar et al., 2014). TRP channels were first described in the fruit flyDrosophila melanogasteras a phototransductiontrpmutant associated with a defect in light-induced calcium entry and a modified response to light (Minke, 1977; Hardie and Minke, 1992). Subsequently members of the TRP family have been identified in vertebrates, and other invertebrates, and in lower eukaryotes such as yeast and fungi. However, so far TRPs or their exact homologs have not been detected in plants (Kumar et al., 2014).

TRPs are classified on the basis of their sequence homology and by the presence of specific signature domains and motifs such as the TRP-domain, TRP-box motifs, ankyrin repeats, etc. (Clapham, 2003). Based on amino acid sequence, homology with other TRP channels, and the presence of specific structural features, the Drosophila TRPshave been divided into seven subfamilies, namely, TRPC (Canoical or Classical), TRPV (Vanilloid-like), TRPM (Melastatin-like), TRPA (Ankyrin), TRPP (Polycystin), TRPML (Mucolipin) and TRPN (No Mechanoreceptor Potential C, NOMPC) (Nilius and Owsianik, 2011). However, the mammalian TRPs have been divided into six subfamilies (the TRPN family does not appear in mammals) (Hartenecket al., 2000; Montell, 2005).

In mammals, TRP channels are ubiquitously expressed in almost all cell types and tissues, albeit at different levels (Kumar et al., 2014). Most of the TRPs are selectively activated by specific ligands and are polymodal in nature (Baez-Nieto, et al., 2011). TRP channels are regulated by multiple stimuli, both physical and chemical and a few members of TRPs are thermosensitive. The complex polymodalregulation of TRP channels by intracellular as well as extracellular components such as, pH, interacting proteins, etc. and the multiple routs of regulation by phosphorylation-dephosphorylation, suggest that these channels integrate multiple signaling events at the plasma membrane (Kumar et al., 2014).

Seven mammalian TRPC channels have been isolated, which are further subdivided by sequence and function into four groups: TRPC1, TRPC2, TRPC3/6/7, and TRPC4/5 (Vazquez, et al., 2004; Liao et al., 2014), although it has been suggested that TRPC1 is dissimilar enough from TRPC4 and TRPC5 that it should comprise its own fourth group (Montell, 2005). All TRPC homologs studied were widely expressed within the central nervous system (CNS) and expression in peripheralnervous system (PNS)was often observed. In spite of this, each type of channel exhibited a different distribution profile (Riccioet al., 2002).

Human TRP3is highly expressed in CNS and smooth and cardiac muscle cells, and likely to play a role in both excitable as well as non-excitable cells, being potentially involved in a wide spectrum of Ca²⁺signalling mechanisms (Li et al., 1999). TRP3 shows multiple potential sites for regulatory phosphorylation in both amino and carboxy termini located in the cytoplasm (Eder et al., 2007). Interactions between different protein-interacting domains in TRP3 channels and a variety of regulatory proteins have been identified that are apparently essential for correct targeting or activation of TRP3 channels (Sinkinset al., 2004).

The Serum- and glucocorticoid-induced protein kinase, SGK (denoted as SGK1), was identified as a novel serine/threonine protein kinase under transcriptional control by serum and glucocorticoids (Webster et al., 1993). The SGK1 gene encodes a 50 kDa protein that is a member of the “AGC” family of serine/threonine protein kinases that includes protein kinases A (PKA),G (PKG), andC (PKC)(Bruhn et al., 2010). SGK1 has two other protein homologues (SGK2 and SGK3), and there are four SGK1 isoforms that are products of alternate translation initiation and can localize to a variety of cellular compartments (Arteagaet al., 2007).

SGK1 is a ubiquitously expressed serine–threonine kinase, highly expressed in the nervous system, playing an important physiological role in CNS in which it regulates different ion channels (Weschet al., 2010). SGK1 has previously been shown to regulate a wide variety of carriers and ion channels (Lang et al., 2006), including the epithelial Ca²⁺ channels TRPV5 (Embark et al., 2004) and TRPV6 (Sopjaniet al., 2010).Furthermore, SGK1 participates in the regulation of renal tubular Na⁺ reabsorption, salt appetite, and thus blood pressure by regulation of renal epithelial Na⁺ channel (ENaC) (Chen et al., 1999).

SGK1 transcription is stimulated by increase in cytosolic Ca²⁺ activity [Ca²⁺]_i (Lang and Stournaras, 2013). SGK1 has been shown to be critically important for the Ca²⁺ entry into mast cells after activation of the IgE receptor (Sobiesiaket al., 2009), an effect mediated by regulation of Ca²⁺ channel 1 (ORAI Calcium Release-Activated Calcium Modulator 1(Orai1)/ Stromal interaction molecule 1 (STIM1)) (Eylensteinet al., 2011). Recent observations revealed a powerful effect of the SGK1 on Orai1 abundance, SOCE, activation and function of platelets (Borstet al., 2012).

Interestingly, SGK contains a proline rich region (PXXP motif, where x denotes any amino acid) in its amino terminal regulatory domainand another protein interaction motif called PDZ domain in its carboxy terminal (O'Keeffe et al., 2013).SGK1 includes three such PXXP motifs which could be involved in protein-protein interactions withproteins containing tryptophan-rich WW motif and thereby potentially modulate its activity (Zhou and Snyder, 2005). The name PDZ is derived from the first three proteins in which these domains were identified: PSD-95 (a 95 kDa protein involved in signaling at the post-synaptic density), DLG (the Drosophila melanogaster Discs Large protein) and ZO-1 (the zonulaoccludens 1 protein involved in maintenance of epithelial polarity) (Harris and Lim, 2001).

The aim of the current study was to investigate whether hTRP3 is regulated by SGK1 and/or the closely related isoforms SGK2, and SGK3. To this end, cRNA encoding wild-type hTRP3 has been injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SGK1, SGK2, and SGK3. The experiments described here were performed by measuring ionic currents from Xenopus oocytes stably expressing hTRP3 using the two-electrode voltage-clamp technique.

MATERIALS AND METHODS

Molecular biology

Plasmid DNA of the human wild-type TRP3 (Zhu et al., 1996; Zhu et al., 1998), of human wild-type SGK1 (Waldeggeret al., 1997), human wild-typeSGK2 (Kobayashi et al., 1999) and human wild-type SGK3 (Kobayashi et al., 1999) were linearized with NotI(Source: Nocardiaotitidis bacteria) and transcribed in vitro with T7 RNA polymerase in the presence of the cap analog m⁷G(5¢)ppp(5¢)G at a concentrationof1mM.TemplatecDNAwasremovedby digestionwithRNase-freeDNaseI.Thecomplementary RNA (cRNA) was purified by phenol/chloroform extraction followed by precipitation with 0.5 volumes 7.5 M ammonium acetate and 2.5 volumes of ethanol to remove unincorporated nucleotides. The integrity of the transcripts was checked by denaturing agarose gel electrophoresis. The complete constructs were sequenced to prove the correct nucleotide exchange and to exclude any additional mutations.

Expression in Xenopus laevis oocytes

StagesV and VI oocytes were harvested from female wild type Xenopuslaevis (Knysna, Rep. South Africa) using previously described procedures (Wagner et al., 2000). Briefly, frogs were anaesthetized by immersion in 0.1% 3-aminobenzoic acid ethyl ester in water. Small pieces of ovary were removed and the incision sutured. Frogs were placed in shallow water until full recovery of reflexes, and subsequently released into the tank. Oocytes were injected with 20ng/25nlcRNA of hTRP3 using a microinjection device (Bachofer, Reutlingen, Germany).In a second step, the oocytes were injected with 25nl water, as a control, or with 7.5ng/25nl water cRNA ofwild type kinases (SGK1, SGK2, and SGK3). All experiments were performed at room temperature (20–24 °C)2-3 days after injection of the respective cRNAs.

Voltage-clamp analysis

In two-electrode voltage-clamp experiments, currents were recorded during a 5-s linear voltage ramp from -150 mV to +50 mV. The intermediate holding potential between the voltage ramps was -50 mV. Data were filtered at 10 Hz and recorded with MacLab digital to analog converter and software for data acquisition and analysis (ADInstruments, Castle Hill, Australia). The bath solution contained 96 mMNaCl, 2 mMKCl, 1 mM MgCl₂, 1mM BaCl₂,10 µM methoxyverapamil, 5 mM HEPES, pH 7.4 with or without 10 mM CaCl₂. Oocytes were kept in modified Barth´s solution containing 88 mMNaCl, 1 mMKCl, 2.4 mM NaHCO₃, 0.8 mM MgSO₄, 0.3 mMCa(NO₃)₂, 0.4 mM CaCl₂ and 5 mM HEPES, pH 7.4 supplemented with 25 µg/ml gentamycin. The final solutions were titrated to the pH indicated using HCl or NaOH. The flow rate of the superfusion was 20 ml/min and a complete exchange of the bath solution was reached within about 10 s. For determination of Ca²⁺ currents (I_Ca), the experiments were performed with Cl^- depleted oocytes (bathed for 24 hours in Cl- free medium) in the absence of extracellular Cl^- in the bath and KCl (3 M) filled agar bridges were used as reference electrodes to minimize liquid junction potentials. In the presence of Cl^- and absence of Cl^- channel inhibitors, the addition of 10 mM CaCl₂ induced an inward current (I_Cl(Ca)) which was created by entry of Ca²⁺ and subsequent activation of Ca²⁺ sensitive Cl^- channels (Hoenderopet al., 1999b; Machaca and Hartzell, 1999). The peak inward current was taken as a measure for hTRP3 activity. I_Cl(Ca) activity is synchronously triggered by the intracellular calcium concentration close to the membrane determined by hTRP3. Thus, I_Cl(Ca) activity mirrors activation and inactivation kinetics of hTRP3(Miledi and Parker, 1984). Expression and currents may vary from batch to batch. Thus, care was taken to make comparisons always within batches.

Statistical analysis

Data are expressed as mean ±s.e.m., where n is the number of oocytes investigated. All experiments were repeated with at least 3 batches of oocytes; in all repetitions qualitatively similar data were obtained. All data were tested for significance using paired or unpaired Student’st-test, and only results with P< 0.05 were considered as statistically significant. The statistical software Origin 8.1 (OriginLab Corp., Northampton, MA) was used to perform all statistical analyses.

RESULTS

Activation of Ca²⁺ sensitive Cl^- channelsin hTRP3-expressing oocytes by cell membrane hyperpolarization.

The current was observed with Xenopus oocytes at 2 days after the injection of hTRP3 cRNA. In hTRP3-expressing oocytes, Ca²⁺ entry through hTRP3 triggered a hyperpolarization-activated inward current by activation of Ca²⁺ sensitive Cl⁻ channels (I_Cl(Ca)) (Fig. 1B). Addition of 10 mM CaCl₂ significantly increased this current (Fig. 1B).

Fig. 1.hTRP3 mediated calcium currents indirectly activate an endogenous chloride conductance (I_Cl(Ca)). A: The cartoon at the top shows the applied two-electrode voltage-clamp protocol. B: Representive original voltage-clamp recording to Ca²⁺ induced Cl^- current (I_Cl(Ca)) from hTRP3-expressing oocytes elicited by linear voltage ramp from -150 mV to +50 mV delivered at 5 sec intervals from a holding potential of -50 mV. Currents were recorded in the presence and absence of 10 mM CaCl₂.

Stimulation of Ca²⁺ induced current by SGK1 in Xenopus oocytes

In the presence of Cl^- the Ca²⁺ entry through hTRP3 stimulated Ca²⁺ sensitive Cl^- channels leading to the appearance of a large Cl^- current (I_Cl(Ca)). As shown in Fig. 2, 10 mMCa²⁺triggered a rapidly activating, slowly and partially inactivating inward current in hTRP3 expressing oocytes with additional expression of SGK1.

Fig. 2. Stimulation of the Ca²⁺ induced Cl^- current (I_Cl(Ca)) in Xenopus oocytes by the combined expression of hTRP3 and SGK1. Current-voltage (I/V) relationships of mean peak currents from Xenopus oocytes before and after application of 10 mM CaCl₂.

Effect of SGK2 and SGK3 on hTRP3 mediated currents in Xenopus oocytes

Further studies have been performed to determine the influence of SGK2 and SGK3 on hTRP3 mediated currents. The peak inward current was taken as a measure for hTRP3 activity. As reported earlier, the entry of Ca²⁺ triggers Ca²⁺ sensitive Cl^- channels (Miledi and Parker, 1984). As shown in figure 3, coexpression of hTRP3 together with SGK1 led to a significant increase of the current induced by addition of 10 mM CaCl₂. In contrast, the current was not increased by coexpression of hTRP3 with either SGK2 or SGK3.

Fig. 3. Stimulation of the Ca²⁺ induced Cl^- current (I_Cl(Ca)) in Xenopus oocytes by the combined expression of hTRP3, SGK1 but not with SGK2 or SGK3. Bar graphs showing the mean peak inward current of hTRP3-expressing oocytes after application of 10 mM CaCl₂. Arithmetic means ± SEM. * indicates significant difference, (*p < 0.05).

DISCUSSION

The primary objective of the current study was to explore the impacts of serum- and glucocorticoid-induced kinases SGK1, SGK2, and SGK3 on hTRP3 Ca²⁺ channel activity.This investigation reveals a powerful effect of the serum- and glucocorticoid-induced kinase SGK1 on SOCE and discloses a completely novel signaling mechanism in the regulation of hTRP3 Ca²⁺ channel activity. This activity has obvious significantinduction by using SGK1, but not by SGK2 or SGK3.The effect of SGK1 on hTRP3 channel activity requires an intact catalytic subunit, wild-type SGK1(Kobayashi and Cohen, 1999;Boehmeret al., 2003).

Importantly, the present investigation reveals that expression of hTRP3Ca²⁺ channels induces a Ca²⁺ entry allowing theintracellular accumulation ofCa²⁺ and generating a Ca²⁺inward current. In the presence of Cl^-, Ca²⁺ influx through hTRP3 Ca²⁺ channels generates further Cl^- inward currents (Hoenderop et al, 1999a) by activation of endogenousCa²⁺activated Cl^- channels in Xenopus oocytes (Callamaras and Parker, 2000).

SGK1 expression is upregulated by glucocorticoids (Webster et al., 1993), aldosterone (Cowling and Birnboim, 2000), cell shrinkage (Waldeggeret al., 1997) and by a wide range of other factors (Lang and Cohen, 2001). Notably, SGK1 is under transcriptional control of Ca²⁺ions inside the cells (Brickleyet al., 2013) and is thus likely to participate in the regulation of calcium homeostasis by regulation of differentcation channels including hTRP3 channels.

SGK1 has previously been shown to compromise Ca²⁺ influx into cells by regulating different Ca²⁺-permeable TRP channels such as TRPV5 (Palmadaet al., 2005) and TRPV6 (Bohmeret al., 2007). Moreover, SGK1 contributes to regulation ofthe epithelial Na⁺/H⁺ exchanger NHE3 (Yun et al., 2002a) and a wide variety of K⁺ channels (Lauferet al., 2009; Lang et al., 2009) such as the voltage gated K⁺ channel Kv1.3 (Gamperet al., 2002) and the renal epithelial K⁺ channel ROMK1 (Yun et al., 2002b).

Activation of K⁺and Ca²⁺ channels is expected to hyperpolarize the cell membrane and thus enhance the driving force for Ca²⁺ entry through several Ca²⁺ channels. SGK1 expression (Klingelet al., 2000; Tarunoet al., 2008) and activity (Imai et al., 2003) are stimulated by increased cytosolic Ca²⁺ activity. Thus, at least in theory, SGK1 could serve as an amplifier of Ca²⁺ entry leading to induction of Ca²⁺ channel Orai1/STIM1 (Eylensteinet al., 2011) and hTRP3 channels activity (Fig. 4).

Fig. 4.Scheme illustrating the SGK1-sensitive regulation of hTRP3 channel activity. SGK1 participates in the regulation of transport proteins in plasma membrane, such as K⁺ and Orai1/STIM1 channels, which have been shown to be SGK1 targets in different cell types.

Further experimental studies are required to confirm a potentially regulatory effect of SGK1 on hTRP3 channel activity. Theseexperiments will be needed to provide detailed information about the molecular mechanism behind SGK1-dependent regulation of hTRP3 channel activity.

CONCLUSIONS

In conclusion, the present observations identify hTRP3as a target of a regulatory mechanism involving the serine/threonine kinase. The SGK1 is a novel transcriptional and powerful regulator of hTRP3 which is at least partially effective through activation of Ca²⁺ entry as well as channel activity. Thus, SGK1-dependent hTRP3 regulations can influence SOCE and activation-dependent Ca²⁺ entry as well as Ca²⁺-dependent mechanisms in brain, heart, and smooth muscle cells.

ACKNOWLEDGEMENTS

The authorgratefully acknowledges all members and professors of the Department of Animal Physiology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt and of Physiology Institute, Faculty of Medicine, EberhardKarls University, Tuebingen, Germany, where the present work was performed, for their constant encouragement and kind help.

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