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Regulation of acid-sensing ion channel 1a function by tissue kallikrein may be through channel cleavage
Neuroscience Letters 490 (2011) 46–51
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Regulation of acid-sensing ion channel 1a function by tissue kallikrein may be through channel cleavage Jingjing Su a , Yuping Tang a , Ling Liu b , Houguang Zhou a , Qiang Dong a,c,∗ a b c
Department of Neurology, Huashan Hospital, Fudan University, Shanghai 200040, China Department of Neurology, Jinling Hospital, Nanjing University School of Medicine, Nanjing 210002, China State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200032, China
a r t i c l e
i n f o
Article history: Received 26 August 2010 Received in revised form 26 November 2010 Accepted 9 December 2010 Keywords: Acid-sensing ion channels Tissue kallikrein Channel cleavage Function Regulatory mechanisms
a b s t r a c t Recently, we have demonstrated that serine protease tissue kallikrein (TK) can protect cortical neurons against ischemia-acidosis/reperfusion-induced injury, and that this effect might be mediated by acidsensing ion channels (ASICs). However, little is known about how TK regulates the function of ASICs. Here we provided evidence that the regulation of ASIC1a function by TK was probably correlated with its cleavage. High concentration of TK (3 M) partially cleaved the extracellular loop of ASIC1a, followed by a marked decrease of LDH release and an increase of cell survival at pH 6.2. Pretreatment with a protease inhibitor aprotinin inhibited the cleavage of ASIC1a and prevented functional regulation by TK. However, the cleavage of ASIC2a, which was not functionally modified by TK, was not observed. Therefore, we propose that the limited proteolysis of extracellular loop within ASIC1a might be one of the potential regulatory mechanisms of ASIC1a function by TK. © 2010 Elsevier Ireland Ltd. All rights reserved.
Acid-sensing ion channels (ASICs) are non-voltage-gated Na+ channels and belong to the members of epithelial Na+ channel (ENaC)/degenerin family, which share a similar topology with an intracytoplasmic N- and C-terminus and have two membrane spanning domains connected by a large extracellular loop. So far, six ASIC subunits including 1a, 1b, 2a, 2b, 3, and 4 have been cloned. ASIC1a, 2a and 2b are expressed abundantly in the peripheral and the central nervous system, whereas the expression of ASIC1b and 3 is restricted to the peripheral nervous system [26]. Of all ASICs, ASIC1a is the most abundantly and ubiquitously expressed in the central nervous system. It is well established that ASICs are transiently activated by a rapid drop in extracellular pH and contribute to neuronal death during cerebral ischemia [9]. ASIC1a begins to open at pH 7.0 and reaches half-maximal activation at pH 6.2 [24]. In contrast, ASIC2a requires the pH values below 5.5 for its activation and its half-maximal activation pH (pH0.5 ) is 4.4 [25]. A variety of studies have demonstrated that the activation of ENaC/degenerin family channels could be regulated by serine proteases. Marin et al. [13] observed that the activatory sites for FMRF-amide and amitriptyline in the extracellular domain of ASIC1a could be removed by trypsin. Neaga et al. [14] found that
∗ Corresponding author at: Department of Neurology, Huashan Hospital, Fudan University, 12 Middle Wulumuqi Road, Shanghai 200040, China. Fax: +86 21 64174579. E-mail addresses: [email protected] (J. Su), [email protected] (Q. Dong). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.12.023
extracellular trypsin could increase the selectivity of ASIC1a for monovalent versus divalent cations. Poirot et al. [20] provided evidence that bi-directional regulation of ASIC1a activation by protease occurred. ENaC, which belongs to the same family as ASICs, is also the target of serine proteases. Previous studies have reported that both extracellular and intracellular proteases can either activate or inactivate ENaC [6,10,22]. Thus, these findings lead us to probe the underlying mechanism by which serine proteases regulate ENaC/degenerin family channels. Vukicevic et al. [23] reported that extracellular trypsin cleaved ASIC1a in the extracellular loop with a similar time course as it changed ASIC1a function, suggesting the regulation of ASIC1a function by trypsin was tightly linked to channel cleavage. Therefore, serine protease influencing ENaC/degenerin family channel function may be through channel cleavage. Tissue kallikrein (TK), a serine protease, is capable of cleaving low molecular weight kininogen to release bradykinin, which can activate bradykinin B2 receptor (B2 R) [8]. Previous reports showed that TK activity in brain can be induced during cerebral ischemia and others [2,7]. Recently, we reported that TK can protect cortical neurons against ischemia-acidosis/reperfusion-induced injury, and that this effect might be mediated by ASICs [11]. However, the precise mechanism about the regulation of ASICs function by TK remains unknown. Since previous studies have demonstrated that incubation of renal cortical membrane fractions from TK−/− mice with TK resulted in the ␥-ENaC cleavage and in vitro addition of TK in mouse cortical collecting ducts significantly increased intracel-
J. Su et al. / Neuroscience Letters 490 (2011) 46–51
lular Na+ concentration [18], we speculate that TK might function as a serine protease to regulate ASICs activation. In this study, we demonstrated that the function of ASIC1a could be regulated by high level of TK, which was probably related to channel cleavage. Moreover, aprotinin could prevent functional regulation and channel cleavage of ASIC1a by TK. TK (brand name is Kailikang) was from Tec pool Bio-Parma Co. (Guangzhou, China). It has been purified from fresh human urine by affinity chromatography with aprotinin coupled to CH-Sepharose and by gel filtration as previously described [17]. It is the active substance of human urinary kallidinogenase and the amino acids of its active center are Asp, His and Ser [12]. Psalmotoxin 1 (PcTX1), a specific ASIC1a blocker, was purchased from Peptides International (Louisville, KY, USA), which is a peptide toxin from the venom of the South American tarantula Psalmopoeus cambridgei. PcTX1 is characterized by the unusual quadruplet Lys25-Arg26-Arg27Arg28, which forms the interaction site of PcTX1 with ASIC1a at the surface of the toxin molecule [4]. Amiloride, a nonspecific ASICs blocker, and aprotinin, a protease inhibitor, were obtained from Sigma (St. Louis, MO, USA). BALB/c mouse were purchased from the Shanghai Institute of the Chinese Academy of Science. All of the procedures complied with Fudan University experimental standards as well as the NIH Guide for the care and use of laboratory animals. Total RNA was prepared from mouse brain with Trizol reagent (Invitrogen). The cDNAs encoding ASIC1a and ASIC2a were amplified by PCR and were cloned into pcDNA3.1/Myc which is a commercial expression vector and is detected conveniently through the recognition of Myc epitope. The Myc epitope in the constructs was located at the C-terminus. CHO and HeLa cells were cultured in Ham’s F12K and DMEM medium, respectively, which was supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37 ◦ C with 5%CO2 . All transfections were carried out using LipofectAMINE2000 according to the manufacturer’s instructions. The cell lines stably expressing ASIC1a and ASIC2a in CHO cells were established as previously described [26]. Immunofluorescence assay for ASICs subcellular localization was performed with anti-Myc antibody (1:500, Sigma) in permeabilized CHO and HeLa cells using a similar technique as previously described [11]. Besides, anti-ASIC1a (1:100, LSBio, LS-C93910) and anti-ASIC2a (1:100, LSBio, LS-C93915) antibodies which were raised against peptide sequences of the extracellular part of ASIC1 and ASIC2, respectively, were also used to detect the localization of ASICs without permeabilizing. Fluorescent labeling was then visualized under a confocal microscope (Leica TCS SP5, Solms, Germany) in 63× objective/10× ocular. TK activity was analyzed as previously described [5]. Cells were incubated with series concentrations of TK (0.03–3 M), PcTX1 (100 ng/ml) or amiloride (100 M) and were treated with low pH solutions (pH 6.2 or 5) according to previous report [11]. For inactivation of TK, TK and aprotinin was preincubated together for 1 h at 37 ◦ C before application and the final concentration of aprotinin was 10 M. Cell injury was determined by measuring the release of lactate dehydrogenase (LDH) in the various treatments using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) as previously described [11]. Cell viability was assessed by WST-8 assay using Cell Counting Kit-8 (CCK-8, Dojindo Laboratories) according to previous report [11]. Western-blot was performed according to Santa Cruz Biotechnology’s protocol. Results were expressed as mean ± SD. Comparisons among groups were made by one-way ANOVA to detect significant difference. p < 0.05 was considered to be statistically significant.
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To detect the expression of mouse ASIC1a and ASIC2a in CHO and HeLa cells transfected with pcDNA3.1/ASIC1a and pcDNA3.1/ASIC2a, respectively, western blot analysis was carried out with anti-Myc antibody. Both ASIC1a and ASIC2a could be detected in the cell extracts with a molecular mass of approximately 64 kDa. However, the obvious band could not be detected in control cells (Fig. 1A and B). To determine the subcellular localization of ASICs in cells, immunofluorescence analysis was performed. The results showed that ASIC1a and ASIC2a were around cell membrane when detected with anti-Myc antibody in permeabilized cells. Anti-ASIC1a and anti-ASIC2a antibodies which recognized extracellular epitopes of ASICs, further revealed that ASIC1a and ASIC2a could be localized on the cell surface in non-permeabilized cells. However, ASICs expression was not obviously observed in pcDNA3.1-transfected cells (Fig. 1C and D). To determine the effects of pH values on TK activity, several pH values were investigated according to previous report [26]. No difference in TK activity was observed between pH 5.0, 6.2, or 7.4 (6.95 ± 0.85, 7.75 ± 0.83, 8.03 ± 0.95 mol/min/mg protein). There was a significant decrease of TK activity when the pH value dropped to 4.4 (4.98 ± 0.76 mol/min/mg protein). Therefore, the effects of TK on the regulation and expression of ASICs were evaluated above pH 5.0. Previous reports have demonstrated that ASIC1a begins to open at pH 7.0 and its pH0.5 is 6.2 [24]. In contrast, ASIC2a channel requires pH values below 5.5 for its activation and its pH0.5 is 4.4 [25]. Therefore, we examined the regulation of ASIC1a and ASIC2a by TK at pH 6.2 and 5.0, respectively, as well as the expression level of ASIC1a and ASIC2a after TK incubation. To observe the effects of ASICs under acidic conditions on cell injury, we tested the LDH release and analyzed the cell survival of ASICs-transfected CHO cells. There was a significant increase of LDH release from ASIC1a-transfected cells at pH 6.2, compared with at pH 7.4. A marked difference of LDH release from ASIC2atransfected cells was also observed between at pH 5.0 and pH 7.4. Specially, pretreatment of PcTX1 or amiloride remarkably suppressed this increase of LDH release from ASIC1a or ASIC2atransfected cells at pH 6.2 or 5.0, respectively (Fig. 2A). Additionally, a cell viability assay showed that acid incubation resulted in the decrease of the cell survival of ASIC1a-transfected cells at pH 6.2 as well as that of ASIC2a-transfected cells at pH 5.0. The ASICs blockers could rescue the cell viability at pH 6.2 or 5.0 (Fig. 2B). However, acid incubation did not markedly increase the LDH release and decrease the cell survival of pcDNA3.1-transfected cells (Fig. 2A and B). These observations suggest that overexpression of ASIC1a and ASIC2a in CHO cells was involved in acid-induced cell injury. To evaluate the effects of TK on the cell injury induced by ASICs, series concentrations of TK were used in various pH conditions. Only high concentration of TK (3 M) showed a significant decrease of LDH release and enhancement of cell survival of ASIC1a-transfected cells at pH 6.2, compared with cells without TK incubation. Additionally, no substantial change occurred in ASIC1atransfected cells with 3 M TK incubation at pH 7.4 (Fig. 3A and C). These results imply that high level of TK could modulate ASIC1a function only when the channel was transiently activated after acid treatment. However, TK had no obvious effect on ASIC2atransfected cells at pH 7.4 or pH 5.0, suggesting that TK could specifically regulate the function of ASIC1a after acid treatment (Fig. 3B and D). Furthermore, various concentrations of TK had no effect on pcDNA3.1-transfected cells at pH 6.2 or pH 5.0. Taken together, we can conclude that there is a certain evidence that TK may exhibit protective effects on cells via ASIC1a. Noticeably, aprotinin, a protease inhibitor, could abolish the effects of 3 M TK on functional changes in ASIC1a-transfected cells at pH 6.2, indicating that the proteolytic activity of TK was required for the regulation of ASIC1a (Fig. 3A and C).
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J. Su et al. / Neuroscience Letters 490 (2011) 46–51
Fig. 1. Western blot analysis for ASICs expression (A and B) and immunofluorescence assay for their subcellular localization (C and D) in CHO and HeLa cells. The lower band with molecular mass of approximately 60 kDa in Panel A was a non-specific one with anti-Myc antibody in CHO cells. (C and D) The expression of ASICs was indicated in green and the nucleus was shown in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
To investigate whether TK had an effect on ASICs cleavage, we analyzed the expression of ASICs in ASIC1a and ASIC2a-transfected cells by western blot analysis with anti-Myc antibody in the presence of TK. Although the lower concentrations of TK exposure (0.03–0.3 M) at pH 6.2 resulted in the appearance of faint bands of
ASIC1a with lower molecular weight, 3 M TK exposure at pH 6.2 led to the appearance of an obvious band about 50 kDa, suggesting that TK could cleave ASIC1a. Notably, pretreatment with aprotinin prevented ASIC1a cleavage by TK. Additionally, the cleavage of ASIC1a by TK (3 M) at pH 7.4 was not observed (Fig. 3E). In con-
Fig. 2. ASIC1a and ASIC2a in CHO cells are involved in cell injury after acid treatments (n = 8 for each group). ***p < 0.001 for pH 6.2 + ASIC1a versus pH 7.4 + ASIC1a, pH 6.2 + ASIC1a versus pH 6.2 + pcDNA3.1, pH 5.0 + ASIC2a versus pH 7.4 + ASIC2a, and pH 5.0 + ASIC2a versus pH 5.0 + pcDNA3.1. ### p < 0.001 for pH 6.2 + PcTX1 or pH 6.2 + amiloride versus pH 6.2 in ASIC1a-transfected cells, and pH 5.0 + amiloride versus pH 5.0 in ASIC2a-transfected cells.
J. Su et al. / Neuroscience Letters 490 (2011) 46–51
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Fig. 3. (A–D) Regulation of ASIC1a function by high concentration of TK in CHO cells at pH 6.2. After acid treatment, only 3 M TK led to significant reduction of LDH release (A) and rise of cell survival (C) in ASIC1a-transfected cells at pH 6.2, which could be prevented by arpotinin. Incubation with various concentrations of TK had no significant effects on cell injury in ASIC2a-transfected cells at the different pH values (B and D) (n = 8 for each group). ***p < 0.001 for pH 6.2 versus pH 7.4, and pH 5.0 versus pH 7.4. ### p < 0.001 for pH 6.2 + PcTX1 versus pH 6.2, and pH 5.0 + amiloride versus pH 5.0. p < 0.001 for pH 6.2 + TK (3 M) versus pH 6.2. (E and F) Selective cleavage of ASIC1a by high concentration of TK in CHO cells at pH 6.2.
trast, no band of ASIC2a with lower molecular mass was detected when ASIC2a-transfected cells exposed to the various concentrations of TK at pH 7.4 or 5.0 (Fig. 3F). These results demonstrate that high concentration of TK could specifically partially cleave ASIC1a only after the channel was transiently activated by extracellular acidification. In this study, we report that high concentration of TK could inhibit the LDH release and enhance the cell survival of ASIC1atransfected cells after acid treatment. TK might regulate the ASIC1a activation through inducing ASIC1a cleavage. Furthermore, pretreatment with a protease inhibitor aprotinin could inhibit the cleavage of ASIC1a and prevent functional regulation by TK. However, TK had not a role in the ASIC2a activation and its cleavage. Our data suggest that the effect of TK on ASIC1a function was probably linked to channel cleavage. First, high concentration of TK at pH 6.2 alleviated cell injury induced by activated ASIC1a and could cleave ASIC1a protein, whereas lower concentrations at pH 6.2 or high concentration of TK at pH 7.4 did not lead to the obvious ASIC1a cleavage and impair its activity. Second, acid-induced
ASIC2a activation was not regulated and ASIC2a channel was not cleaved by TK. Third, pretreatment with aprotinin prevented all functional regulation and channel cleavage of ASIC1a by TK, suggesting that the enzyme activity of TK might be necessary for these effects. However, Andreasen et al. [1] observed that mouse channelactivating protease (mCAP-1) catalytically inactive mutants were still able to fully activate ENaC, but not mCAP2 and mCAP3, suggesting that noncatalytic mechanisms were involved in this activation processing. They proposed that a possible interaction mechanism might be involved in ENaC activation. The mCAP-1 might interact with protease inhibitors, thereby squelching their activity on other proteases that can activate ENaC through the catalytic pathway. In our study, TK had been purified from fresh human urine by affinity chromatography with aprotinin coupled to CH-Sepharose and yielded an overall recovery of more than 75% in TK activity in a 36 h period [17]. Therefore, aprotinin binding to TK would likely also prevent cleavage-independent effects by sequestering TK. Given that the cleaved ASIC1a had a molecular mass of 50 kDa and the Myc epitope was located at the C-terminus of ASIC1a, we
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J. Su et al. / Neuroscience Letters 490 (2011) 46–51
presume the cleavage of ASIC1a might occur near the N-terminal part of extracellular loop and subsequently change the conformation of extracellular loop, thereby inducing the loss of channel activation [23]. Previous studies reported that short substrates which were cleaved after the Phe–Arg (FR) and Phe–Phe (FF) pairs were preferable for TK hydrolysis, and the limited proteolytic activity of TK was also extended to its arginylhydrolase function [19]. As shown in supplemental Fig. 1, there were several FR and FF pairs in the extramembrane parts of ASIC1a which might function as the recognition site of TK activity. However, the accurate cleavage site remains to be identified. Furthermore, previous studies have shown that the efficiency of the substrate hydrolysis by TK was also highly dependent on secondary structure of substrates rather than absolute sequence [19]. Our results showed that TK could cleave ASIC1a at pH 6.2 but not at 7.4 and however ASIC2a was not cleaved by TK. Therefore, it was presumed that a conformational change in ASIC1a may occur at pH 6.2, which rendered the cleavage site either largely accessible to TK or increased its reactivity toward extracellular TK and led to ASIC1a cleavage [23]. Similarly, the conformational change in ASIC2a did not result in the exposure of the cleavage site and therefore, ASIC2a could not be cleaved though three potential TK-sensitive sites in the extracellular loop of ASIC2a. The reasons for these observations might be that conditions used in this study are not optimal for such a cleavage reaction in ASIC2a. It is clear that ASICs are transiently activated by a rapid drop in extracellular pH [9] and desensitized rapidly after continuous acid [3]. Our results revealed that TK regulated ASIC1a activation and cleaved it at pH 6.2, but not at 7.4. Therefore, the effects of TK on ASIC1a channel occurred when the channel was desensitized after transient activation. Interestingly, Vukicevic et al. [23] showed opposite observations that trypsin cleaved closed ASIC1a at pH 7.5 but not desensitized ASIC1a. The possible explanations for this discrepancy are that the molecular conformation and cleavage site exposure during inactivation and desensitization are not identical. It has been shown that tissue acidosis is a well established feature of cerebral ischemia and plays a critical role in brain injury [16]. During cerebral ischemia tissue pH falls to 6.5–6.0 and can fall even below 6.0 during severe ischemia, which is sufficient to activate ASIC1a channel with a pH0.5 at 6.2 [24,15]. In the present study, we found that TK modified ASIC1a activation and cleaved it at pH 6.2. However, TK had not an effect on the ASIC1a activation at pH 7.4. The results demonstrate that TK might have an inhibitory effect on the ASIC1a activation during cerebral ischemia. Our previous studies showed that TK activity undertook a rapid induction in ischemic brain of rats with middle cerebral artery occlusion (MCAO) for 90 min. TK activity reached its maximum by 4.48-fold at 2 h after MCAO and thereafter returned to baseline level at 24 h (unpublished data). To investigate the potential role of TK in ischemic brain, we also constructed the Protein Transduction Domain-TK (PTD-TK) and observed its influence on cultured neurons with oxygen and glucose deprivation (OGD) in vitro and ischemic rats with MCAO. The results revealed that PTD-TK had neuroprotective effects on neurons and ischemic brain [21]. Despite the induction in TK protective activity during cerebral ischemia, its effects are still transient and not enough to offer more neuroprotection against stroke. Here, we observed that only high level of TK may exhibit its nonreceptor-mediated effects and provide the protection against cell injury induced by ASIC1a after acid treatment, which was consistent with our previous studies that TK remained high level only for a short time and its effects were still transient. Therefore, exogenous administration of high level of TK might be required to confer its neuroprotective effects during cerebral ischemia. In conclusion, the present work has established that high concentration of TK cleaved ASIC1a and led to substantial functional
changes of ASIC1a in CHO cells, which might exhibit a potential regulatory mechanisms of ASIC1a function by TK during cerebral ischemia. We also speculate that ASIC1a cleavage by TK might change its properties, but not render it non-functional. These findings raised the possibility that high level of TK might have more value in the treatment of acute ischemic stroke. Acknowledgement This work was supported by Innovation Fund for graduates in Fudan University of Shanghai. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2010.12.023. References [1] D. Andreasen, G. Vuagniaux, N. Fowler-Jaeger, E. Hummler, B.C. Rossier, Activation of epithelial sodium channels by mouse channel activating proteases (mCAP) expressed in Xenopus oocytes requires catalytic activity of mCAP3 and mCAP2 but not mCAP1, J. Am. Soc. Nephrol. 17 (2006) 968–976. [2] J. Chao, C. Woodley, L. Chao, H.S. Margolius, Identification of tissue kallikrein in brain and in the cell-free translation product encoded by brain mRNA, J. Biol. Chem. 258 (1983) 15173–15178. [3] T. Coric, P. Zhang, N. Todorovic, C.M. Canessa, The extracellular domain determines the kinetics of desensitization in acid-sensitive ion channel 1, J. Biol. Chem. 278 (2003) 45240–45247. [4] P. Escoubas, J.R. De Weille, A. Lecoq, S. Diochot, R. Waldmann, G. Champigny, D. Moinier, A. Ménez, M. Lazdunski, Isolation of a tarantula toxin specific for a class of proton-gated Na+ channels, J. Biol. Chem. 275 (2000) 25116–25121. [5] L. Gao, L. Chao, J. Chao, A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: activation of proteinase-activated receptor 1 and epidermal growth factor receptor, Exp. Cell. Res. 316 (2009) 376–389. [6] H. Garty, I.S. Edelman, Amiloride-sensitive trypsinization of apical sodium channels. Analysis of hormonal regulation of sodium transport in toad bladder, J. Gen. Physiol. 81 (1983) 785–803. [7] M.B. Gingrich, S.F. Traynelis, Serine proteases and brain damage-is there a link? Trends Neurosci. 23 (2000) 399–407. [8] C. Hecquet, F. Tan, B.M. Marcic, E.G. Erdös, Human bradykinin B(2) receptor is activated by kallikrein and other serine proteases, Mol. Pharmacol. 58 (2000) 828–836. [9] O. Krishtal, The ASICs: signaling molecules? Modulators? Trends Neurosci. 26 (2003) 477–483. [10] S.A. Lewis, W.P. Alles, Urinary kallikrein: a physiological regulator of epithelial Na+ absorption, Proc. Natl. Acad. Sci. U.S.A. 83 (1986) 5345–5348. [11] L. Liu, R. Zhang, K. Liu, H. Zhou, X. Yang, X. Liu, M. Tang, J. Su, Q. Dong, Tissue kallikrein protects cortical neurons against in vitro ischemiaacidosis/reperfusion-induced injury through the ERK1/2 pathway, Exp. Neurol. 219 (2009) 453–465. [12] H.S. Lu, F.K. Lin, L. Chao, J. Chao, Human urinary kallikrein. Complete amino acid sequence and sites of glycosylation, Int. J. Pept. Protein Res. 33 (1989) 237–249. [13] A.N. Marin, C.M. Prica, B.P. Amuzescu, E.I. Neaga, M.L. Flonta, ASIC1a activation by amitriptyline and FMRF-amide is removed by serine proteases, Channels (Austin) 2 (2008) 419–428. [14] E. Neaga, B. Amuzescu, C. Dinu, B. Macri, F. Pena, M.L. Flonta, Extracellular trypsin increases ASIC1a selectivity for monovalent versus divalent cations, J. Neurosci. Methods 144 (2005) 241–248. [15] M. Nedergaard, R.P. Kraig, J. Tanabe, W.A. Pulsinelli, Dynamics of interstitial and intracellular pH in evolving brain infarct, Am. J. Physiol. 260 (1991) R581–588. [16] E.M. Nemoto, S. Frinak, Brain tissue pH after global brain ischemia and barbiturate loading in rats, Stroke 12 (1981) 77–82. [17] O.K. ole-MoiYoi, J. Spragg, K.F. Austen, Structural studies of human urinary kallikrein (urokallikrein), Proc. Natl. Acad. Sci. U.S.A. 76 (1979) 3121–3125. [18] N. Picard, D. Eladari, S. El Moghrabi, C. Planès, S. Bourgeois, P. Houillier, Q. Wang, M. Burnier, G. Deschenes, M.A. Knepper, P. Meneton, R. Chambrey, Defective ENaC processing and function in tissue kallikrein-deficient mice, J. Biol. Chem. 283 (2008) 4602–4611. [19] D.C. Pimenta, J. Chao, L. Chao, M.A. Juliano, L. Juliano, Specificity of human tissue kallikrein towards substrates containing Phe–Phe pair of amino acids, Biochem. J. 339 (1999) 473–479. [20] O. Poirot, M. Vukicevic, A. Boesch, S. Kellenberger, Selective regulation of acid-sensing ion channel 1 by serine proteases, J. Biol. Chem. 279 (2004) 38448–38457. [21] Y. Tang, Y. Shao, J. Su, H. Zhou, L. Liu, H. Ren, Q. Dong, The protein therapy of kallikrein in cerebral ischemic reperfusion injury, Curr. Med. Chem. 16 (2009) 4502–4510. [22] V. Vallet, A. Chraibi, H.P. Gaeggeler, J.D. Horisberger, B.C. Rossier, An epithelial serine protease activates the amiloride-sensitive sodium channel, Nature 389 (1997) 607–610.
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Regulation of acid-sensing ion channel 1a function by tissue kallikrein may be through channel cleavage Jingjing Su a , Yuping Tang a , Ling Liu b , Houguang Zhou a , Qiang Dong a,c,∗ a b c
Department of Neurology, Huashan Hospital, Fudan University, Shanghai 200040, China Department of Neurology, Jinling Hospital, Nanjing University School of Medicine, Nanjing 210002, China State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200032, China
a r t i c l e
i n f o
Article history: Received 26 August 2010 Received in revised form 26 November 2010 Accepted 9 December 2010 Keywords: Acid-sensing ion channels Tissue kallikrein Channel cleavage Function Regulatory mechanisms
a b s t r a c t Recently, we have demonstrated that serine protease tissue kallikrein (TK) can protect cortical neurons against ischemia-acidosis/reperfusion-induced injury, and that this effect might be mediated by acidsensing ion channels (ASICs). However, little is known about how TK regulates the function of ASICs. Here we provided evidence that the regulation of ASIC1a function by TK was probably correlated with its cleavage. High concentration of TK (3 M) partially cleaved the extracellular loop of ASIC1a, followed by a marked decrease of LDH release and an increase of cell survival at pH 6.2. Pretreatment with a protease inhibitor aprotinin inhibited the cleavage of ASIC1a and prevented functional regulation by TK. However, the cleavage of ASIC2a, which was not functionally modified by TK, was not observed. Therefore, we propose that the limited proteolysis of extracellular loop within ASIC1a might be one of the potential regulatory mechanisms of ASIC1a function by TK. © 2010 Elsevier Ireland Ltd. All rights reserved.
Acid-sensing ion channels (ASICs) are non-voltage-gated Na+ channels and belong to the members of epithelial Na+ channel (ENaC)/degenerin family, which share a similar topology with an intracytoplasmic N- and C-terminus and have two membrane spanning domains connected by a large extracellular loop. So far, six ASIC subunits including 1a, 1b, 2a, 2b, 3, and 4 have been cloned. ASIC1a, 2a and 2b are expressed abundantly in the peripheral and the central nervous system, whereas the expression of ASIC1b and 3 is restricted to the peripheral nervous system [26]. Of all ASICs, ASIC1a is the most abundantly and ubiquitously expressed in the central nervous system. It is well established that ASICs are transiently activated by a rapid drop in extracellular pH and contribute to neuronal death during cerebral ischemia [9]. ASIC1a begins to open at pH 7.0 and reaches half-maximal activation at pH 6.2 [24]. In contrast, ASIC2a requires the pH values below 5.5 for its activation and its half-maximal activation pH (pH0.5 ) is 4.4 [25]. A variety of studies have demonstrated that the activation of ENaC/degenerin family channels could be regulated by serine proteases. Marin et al. [13] observed that the activatory sites for FMRF-amide and amitriptyline in the extracellular domain of ASIC1a could be removed by trypsin. Neaga et al. [14] found that
∗ Corresponding author at: Department of Neurology, Huashan Hospital, Fudan University, 12 Middle Wulumuqi Road, Shanghai 200040, China. Fax: +86 21 64174579. E-mail addresses: [email protected] (J. Su), [email protected] (Q. Dong). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.12.023
extracellular trypsin could increase the selectivity of ASIC1a for monovalent versus divalent cations. Poirot et al. [20] provided evidence that bi-directional regulation of ASIC1a activation by protease occurred. ENaC, which belongs to the same family as ASICs, is also the target of serine proteases. Previous studies have reported that both extracellular and intracellular proteases can either activate or inactivate ENaC [6,10,22]. Thus, these findings lead us to probe the underlying mechanism by which serine proteases regulate ENaC/degenerin family channels. Vukicevic et al. [23] reported that extracellular trypsin cleaved ASIC1a in the extracellular loop with a similar time course as it changed ASIC1a function, suggesting the regulation of ASIC1a function by trypsin was tightly linked to channel cleavage. Therefore, serine protease influencing ENaC/degenerin family channel function may be through channel cleavage. Tissue kallikrein (TK), a serine protease, is capable of cleaving low molecular weight kininogen to release bradykinin, which can activate bradykinin B2 receptor (B2 R) [8]. Previous reports showed that TK activity in brain can be induced during cerebral ischemia and others [2,7]. Recently, we reported that TK can protect cortical neurons against ischemia-acidosis/reperfusion-induced injury, and that this effect might be mediated by ASICs [11]. However, the precise mechanism about the regulation of ASICs function by TK remains unknown. Since previous studies have demonstrated that incubation of renal cortical membrane fractions from TK−/− mice with TK resulted in the ␥-ENaC cleavage and in vitro addition of TK in mouse cortical collecting ducts significantly increased intracel-
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lular Na+ concentration [18], we speculate that TK might function as a serine protease to regulate ASICs activation. In this study, we demonstrated that the function of ASIC1a could be regulated by high level of TK, which was probably related to channel cleavage. Moreover, aprotinin could prevent functional regulation and channel cleavage of ASIC1a by TK. TK (brand name is Kailikang) was from Tec pool Bio-Parma Co. (Guangzhou, China). It has been purified from fresh human urine by affinity chromatography with aprotinin coupled to CH-Sepharose and by gel filtration as previously described [17]. It is the active substance of human urinary kallidinogenase and the amino acids of its active center are Asp, His and Ser [12]. Psalmotoxin 1 (PcTX1), a specific ASIC1a blocker, was purchased from Peptides International (Louisville, KY, USA), which is a peptide toxin from the venom of the South American tarantula Psalmopoeus cambridgei. PcTX1 is characterized by the unusual quadruplet Lys25-Arg26-Arg27Arg28, which forms the interaction site of PcTX1 with ASIC1a at the surface of the toxin molecule [4]. Amiloride, a nonspecific ASICs blocker, and aprotinin, a protease inhibitor, were obtained from Sigma (St. Louis, MO, USA). BALB/c mouse were purchased from the Shanghai Institute of the Chinese Academy of Science. All of the procedures complied with Fudan University experimental standards as well as the NIH Guide for the care and use of laboratory animals. Total RNA was prepared from mouse brain with Trizol reagent (Invitrogen). The cDNAs encoding ASIC1a and ASIC2a were amplified by PCR and were cloned into pcDNA3.1/Myc which is a commercial expression vector and is detected conveniently through the recognition of Myc epitope. The Myc epitope in the constructs was located at the C-terminus. CHO and HeLa cells were cultured in Ham’s F12K and DMEM medium, respectively, which was supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37 ◦ C with 5%CO2 . All transfections were carried out using LipofectAMINE2000 according to the manufacturer’s instructions. The cell lines stably expressing ASIC1a and ASIC2a in CHO cells were established as previously described [26]. Immunofluorescence assay for ASICs subcellular localization was performed with anti-Myc antibody (1:500, Sigma) in permeabilized CHO and HeLa cells using a similar technique as previously described [11]. Besides, anti-ASIC1a (1:100, LSBio, LS-C93910) and anti-ASIC2a (1:100, LSBio, LS-C93915) antibodies which were raised against peptide sequences of the extracellular part of ASIC1 and ASIC2, respectively, were also used to detect the localization of ASICs without permeabilizing. Fluorescent labeling was then visualized under a confocal microscope (Leica TCS SP5, Solms, Germany) in 63× objective/10× ocular. TK activity was analyzed as previously described [5]. Cells were incubated with series concentrations of TK (0.03–3 M), PcTX1 (100 ng/ml) or amiloride (100 M) and were treated with low pH solutions (pH 6.2 or 5) according to previous report [11]. For inactivation of TK, TK and aprotinin was preincubated together for 1 h at 37 ◦ C before application and the final concentration of aprotinin was 10 M. Cell injury was determined by measuring the release of lactate dehydrogenase (LDH) in the various treatments using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) as previously described [11]. Cell viability was assessed by WST-8 assay using Cell Counting Kit-8 (CCK-8, Dojindo Laboratories) according to previous report [11]. Western-blot was performed according to Santa Cruz Biotechnology’s protocol. Results were expressed as mean ± SD. Comparisons among groups were made by one-way ANOVA to detect significant difference. p < 0.05 was considered to be statistically significant.
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To detect the expression of mouse ASIC1a and ASIC2a in CHO and HeLa cells transfected with pcDNA3.1/ASIC1a and pcDNA3.1/ASIC2a, respectively, western blot analysis was carried out with anti-Myc antibody. Both ASIC1a and ASIC2a could be detected in the cell extracts with a molecular mass of approximately 64 kDa. However, the obvious band could not be detected in control cells (Fig. 1A and B). To determine the subcellular localization of ASICs in cells, immunofluorescence analysis was performed. The results showed that ASIC1a and ASIC2a were around cell membrane when detected with anti-Myc antibody in permeabilized cells. Anti-ASIC1a and anti-ASIC2a antibodies which recognized extracellular epitopes of ASICs, further revealed that ASIC1a and ASIC2a could be localized on the cell surface in non-permeabilized cells. However, ASICs expression was not obviously observed in pcDNA3.1-transfected cells (Fig. 1C and D). To determine the effects of pH values on TK activity, several pH values were investigated according to previous report [26]. No difference in TK activity was observed between pH 5.0, 6.2, or 7.4 (6.95 ± 0.85, 7.75 ± 0.83, 8.03 ± 0.95 mol/min/mg protein). There was a significant decrease of TK activity when the pH value dropped to 4.4 (4.98 ± 0.76 mol/min/mg protein). Therefore, the effects of TK on the regulation and expression of ASICs were evaluated above pH 5.0. Previous reports have demonstrated that ASIC1a begins to open at pH 7.0 and its pH0.5 is 6.2 [24]. In contrast, ASIC2a channel requires pH values below 5.5 for its activation and its pH0.5 is 4.4 [25]. Therefore, we examined the regulation of ASIC1a and ASIC2a by TK at pH 6.2 and 5.0, respectively, as well as the expression level of ASIC1a and ASIC2a after TK incubation. To observe the effects of ASICs under acidic conditions on cell injury, we tested the LDH release and analyzed the cell survival of ASICs-transfected CHO cells. There was a significant increase of LDH release from ASIC1a-transfected cells at pH 6.2, compared with at pH 7.4. A marked difference of LDH release from ASIC2atransfected cells was also observed between at pH 5.0 and pH 7.4. Specially, pretreatment of PcTX1 or amiloride remarkably suppressed this increase of LDH release from ASIC1a or ASIC2atransfected cells at pH 6.2 or 5.0, respectively (Fig. 2A). Additionally, a cell viability assay showed that acid incubation resulted in the decrease of the cell survival of ASIC1a-transfected cells at pH 6.2 as well as that of ASIC2a-transfected cells at pH 5.0. The ASICs blockers could rescue the cell viability at pH 6.2 or 5.0 (Fig. 2B). However, acid incubation did not markedly increase the LDH release and decrease the cell survival of pcDNA3.1-transfected cells (Fig. 2A and B). These observations suggest that overexpression of ASIC1a and ASIC2a in CHO cells was involved in acid-induced cell injury. To evaluate the effects of TK on the cell injury induced by ASICs, series concentrations of TK were used in various pH conditions. Only high concentration of TK (3 M) showed a significant decrease of LDH release and enhancement of cell survival of ASIC1a-transfected cells at pH 6.2, compared with cells without TK incubation. Additionally, no substantial change occurred in ASIC1atransfected cells with 3 M TK incubation at pH 7.4 (Fig. 3A and C). These results imply that high level of TK could modulate ASIC1a function only when the channel was transiently activated after acid treatment. However, TK had no obvious effect on ASIC2atransfected cells at pH 7.4 or pH 5.0, suggesting that TK could specifically regulate the function of ASIC1a after acid treatment (Fig. 3B and D). Furthermore, various concentrations of TK had no effect on pcDNA3.1-transfected cells at pH 6.2 or pH 5.0. Taken together, we can conclude that there is a certain evidence that TK may exhibit protective effects on cells via ASIC1a. Noticeably, aprotinin, a protease inhibitor, could abolish the effects of 3 M TK on functional changes in ASIC1a-transfected cells at pH 6.2, indicating that the proteolytic activity of TK was required for the regulation of ASIC1a (Fig. 3A and C).
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Fig. 1. Western blot analysis for ASICs expression (A and B) and immunofluorescence assay for their subcellular localization (C and D) in CHO and HeLa cells. The lower band with molecular mass of approximately 60 kDa in Panel A was a non-specific one with anti-Myc antibody in CHO cells. (C and D) The expression of ASICs was indicated in green and the nucleus was shown in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
To investigate whether TK had an effect on ASICs cleavage, we analyzed the expression of ASICs in ASIC1a and ASIC2a-transfected cells by western blot analysis with anti-Myc antibody in the presence of TK. Although the lower concentrations of TK exposure (0.03–0.3 M) at pH 6.2 resulted in the appearance of faint bands of
ASIC1a with lower molecular weight, 3 M TK exposure at pH 6.2 led to the appearance of an obvious band about 50 kDa, suggesting that TK could cleave ASIC1a. Notably, pretreatment with aprotinin prevented ASIC1a cleavage by TK. Additionally, the cleavage of ASIC1a by TK (3 M) at pH 7.4 was not observed (Fig. 3E). In con-
Fig. 2. ASIC1a and ASIC2a in CHO cells are involved in cell injury after acid treatments (n = 8 for each group). ***p < 0.001 for pH 6.2 + ASIC1a versus pH 7.4 + ASIC1a, pH 6.2 + ASIC1a versus pH 6.2 + pcDNA3.1, pH 5.0 + ASIC2a versus pH 7.4 + ASIC2a, and pH 5.0 + ASIC2a versus pH 5.0 + pcDNA3.1. ### p < 0.001 for pH 6.2 + PcTX1 or pH 6.2 + amiloride versus pH 6.2 in ASIC1a-transfected cells, and pH 5.0 + amiloride versus pH 5.0 in ASIC2a-transfected cells.
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Fig. 3. (A–D) Regulation of ASIC1a function by high concentration of TK in CHO cells at pH 6.2. After acid treatment, only 3 M TK led to significant reduction of LDH release (A) and rise of cell survival (C) in ASIC1a-transfected cells at pH 6.2, which could be prevented by arpotinin. Incubation with various concentrations of TK had no significant effects on cell injury in ASIC2a-transfected cells at the different pH values (B and D) (n = 8 for each group). ***p < 0.001 for pH 6.2 versus pH 7.4, and pH 5.0 versus pH 7.4. ### p < 0.001 for pH 6.2 + PcTX1 versus pH 6.2, and pH 5.0 + amiloride versus pH 5.0. p < 0.001 for pH 6.2 + TK (3 M) versus pH 6.2. (E and F) Selective cleavage of ASIC1a by high concentration of TK in CHO cells at pH 6.2.
trast, no band of ASIC2a with lower molecular mass was detected when ASIC2a-transfected cells exposed to the various concentrations of TK at pH 7.4 or 5.0 (Fig. 3F). These results demonstrate that high concentration of TK could specifically partially cleave ASIC1a only after the channel was transiently activated by extracellular acidification. In this study, we report that high concentration of TK could inhibit the LDH release and enhance the cell survival of ASIC1atransfected cells after acid treatment. TK might regulate the ASIC1a activation through inducing ASIC1a cleavage. Furthermore, pretreatment with a protease inhibitor aprotinin could inhibit the cleavage of ASIC1a and prevent functional regulation by TK. However, TK had not a role in the ASIC2a activation and its cleavage. Our data suggest that the effect of TK on ASIC1a function was probably linked to channel cleavage. First, high concentration of TK at pH 6.2 alleviated cell injury induced by activated ASIC1a and could cleave ASIC1a protein, whereas lower concentrations at pH 6.2 or high concentration of TK at pH 7.4 did not lead to the obvious ASIC1a cleavage and impair its activity. Second, acid-induced
ASIC2a activation was not regulated and ASIC2a channel was not cleaved by TK. Third, pretreatment with aprotinin prevented all functional regulation and channel cleavage of ASIC1a by TK, suggesting that the enzyme activity of TK might be necessary for these effects. However, Andreasen et al. [1] observed that mouse channelactivating protease (mCAP-1) catalytically inactive mutants were still able to fully activate ENaC, but not mCAP2 and mCAP3, suggesting that noncatalytic mechanisms were involved in this activation processing. They proposed that a possible interaction mechanism might be involved in ENaC activation. The mCAP-1 might interact with protease inhibitors, thereby squelching their activity on other proteases that can activate ENaC through the catalytic pathway. In our study, TK had been purified from fresh human urine by affinity chromatography with aprotinin coupled to CH-Sepharose and yielded an overall recovery of more than 75% in TK activity in a 36 h period [17]. Therefore, aprotinin binding to TK would likely also prevent cleavage-independent effects by sequestering TK. Given that the cleaved ASIC1a had a molecular mass of 50 kDa and the Myc epitope was located at the C-terminus of ASIC1a, we
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presume the cleavage of ASIC1a might occur near the N-terminal part of extracellular loop and subsequently change the conformation of extracellular loop, thereby inducing the loss of channel activation [23]. Previous studies reported that short substrates which were cleaved after the Phe–Arg (FR) and Phe–Phe (FF) pairs were preferable for TK hydrolysis, and the limited proteolytic activity of TK was also extended to its arginylhydrolase function [19]. As shown in supplemental Fig. 1, there were several FR and FF pairs in the extramembrane parts of ASIC1a which might function as the recognition site of TK activity. However, the accurate cleavage site remains to be identified. Furthermore, previous studies have shown that the efficiency of the substrate hydrolysis by TK was also highly dependent on secondary structure of substrates rather than absolute sequence [19]. Our results showed that TK could cleave ASIC1a at pH 6.2 but not at 7.4 and however ASIC2a was not cleaved by TK. Therefore, it was presumed that a conformational change in ASIC1a may occur at pH 6.2, which rendered the cleavage site either largely accessible to TK or increased its reactivity toward extracellular TK and led to ASIC1a cleavage [23]. Similarly, the conformational change in ASIC2a did not result in the exposure of the cleavage site and therefore, ASIC2a could not be cleaved though three potential TK-sensitive sites in the extracellular loop of ASIC2a. The reasons for these observations might be that conditions used in this study are not optimal for such a cleavage reaction in ASIC2a. It is clear that ASICs are transiently activated by a rapid drop in extracellular pH [9] and desensitized rapidly after continuous acid [3]. Our results revealed that TK regulated ASIC1a activation and cleaved it at pH 6.2, but not at 7.4. Therefore, the effects of TK on ASIC1a channel occurred when the channel was desensitized after transient activation. Interestingly, Vukicevic et al. [23] showed opposite observations that trypsin cleaved closed ASIC1a at pH 7.5 but not desensitized ASIC1a. The possible explanations for this discrepancy are that the molecular conformation and cleavage site exposure during inactivation and desensitization are not identical. It has been shown that tissue acidosis is a well established feature of cerebral ischemia and plays a critical role in brain injury [16]. During cerebral ischemia tissue pH falls to 6.5–6.0 and can fall even below 6.0 during severe ischemia, which is sufficient to activate ASIC1a channel with a pH0.5 at 6.2 [24,15]. In the present study, we found that TK modified ASIC1a activation and cleaved it at pH 6.2. However, TK had not an effect on the ASIC1a activation at pH 7.4. The results demonstrate that TK might have an inhibitory effect on the ASIC1a activation during cerebral ischemia. Our previous studies showed that TK activity undertook a rapid induction in ischemic brain of rats with middle cerebral artery occlusion (MCAO) for 90 min. TK activity reached its maximum by 4.48-fold at 2 h after MCAO and thereafter returned to baseline level at 24 h (unpublished data). To investigate the potential role of TK in ischemic brain, we also constructed the Protein Transduction Domain-TK (PTD-TK) and observed its influence on cultured neurons with oxygen and glucose deprivation (OGD) in vitro and ischemic rats with MCAO. The results revealed that PTD-TK had neuroprotective effects on neurons and ischemic brain [21]. Despite the induction in TK protective activity during cerebral ischemia, its effects are still transient and not enough to offer more neuroprotection against stroke. Here, we observed that only high level of TK may exhibit its nonreceptor-mediated effects and provide the protection against cell injury induced by ASIC1a after acid treatment, which was consistent with our previous studies that TK remained high level only for a short time and its effects were still transient. Therefore, exogenous administration of high level of TK might be required to confer its neuroprotective effects during cerebral ischemia. In conclusion, the present work has established that high concentration of TK cleaved ASIC1a and led to substantial functional
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