{"created":"2023-06-20T13:21:05.923582+00:00","id":1191,"links":{},"metadata":{"_buckets":{"deposit":"8deeda64-412a-4d7e-a26d-62973b2c7f61"},"_deposit":{"created_by":1,"id":"1191","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"1191"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00001191","sets":["2:430:22"]},"author_link":["0","0","0"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"TOYCHIEV, Abduqodir Hodjiakbarovich"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"トイチェフ, アブド"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2007-09-28"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"The maxi-anion channel is a voltage-dependent, large-conductance anion selective channel. The maxi- anion channels express in a large variety of cell types. Roles of the maxi-anion channel in cell volume regulation, programmed cell death, ischemia and purinergic signaling have been well documented. Its main physiological function is considered as a pathway for the regulated release of ATP and glutamate. <br /> The maxi-anion channel has been extensively characterized with respect to biophysical and pharmacological properties, although its molecular identity is unknown. In spite of some progress in recent years, the mechanisms by which maxi-anion channel is regulated remain incompletely understood. Hence, the purpose of his study was to reveal the activation mechanisms of the maxi-anion channel. Also, He tested a hypothesis of TTHY1 as a molecular identity of the maxi-anion channel.<br />　　　In the present work, He studied excision-induced activation maxi-anion channel by patch-clamp in the inside-out mode. He demonstrated that excision-activated maxi-anion channels in mammary C127 cells show similar biophysical profiles in artificially designed intracellular solution as previously reported for this cell line under different conditions. The biophysical profile includes a large single-channel conductance of ~365 pS, a linear current-to-voltage relationship, time- and voltage-dependent inactivation at higher voltages and anionic selectivity. Addition of the MgATP into the intracellular solution at a physiological level (1 mM) completely abolished excision-induced activation  of the maxi-anion channel in inside-out patches. In contrast, the non-hydrolysable analog of ATP,  ANP-PNP, failed to suppress the maxi-anion channel activation. Based on these results, He supposed that the excised membrane patches retained auxiliary proteins including kinases and phosphatases, and that under ATP-free conditions, phosphatase activities dominated and caused the channel opening whereas in the presence of Mg-ATP the channel protein remained phosphorylated and stayed in the inactive closed state. Therefore, He next addressed the following question: what kind of phosphatase is involved in excision-induced activation of the maxi-anion channel? First He tested the effects of Ser/Thr phosphatase inhibitors (10-100 nM okadaic acid, 1 μM cyclosporine A and 1 μM FK520) on excision-induced activation of the maxi-anion channel. However, neither one had any significant effect on the channel activation. In contrast, broad-spectrum tyrosine phosphatase inhibitors (TPhIC 1%; 1 mM orthovanadate; 0.1 mM dephostatin and 0.1 mM p-bromoteramisole) markedly suppressed the maxi-anion channel activation. These results strongly suggest an involvement of some protein tyrosine,  rather than Ser/Thr, phosphatase in the maxi-anion channel activation. In order to strengthen this inference on the role of tyrosine phosphorylation/dephosphorylation process in the maxi-anion channel activation, He next performed the experiments using inhibitors of protein kinases. The rationale was that in the experimental conditions favoring the phosphorylation process, protein kinase inhibitors should be able to restore the channel activity from its inactive state. In the presence of 1 mM MgATP,  broad-spectrum serine/treonine kinase inhibitor, H7, had no significant effect on the channel activation,  but, in contrast, two tyrosine kinase inhibitors, AG18 and genestein, caused robust activation of the maxi-anion channel over in the presence of MgATP. These results provide independent evidence that tyrosine, but not Ser/Thr, phosphorylation is involved in the inhibition of the maxi-anion channel in C127 cells.<br />　　　Which protein tyrosine phosphatase (PTP) is responsible for the maxi-anion channel activation? To identify the specific type of PTP, He tested available inhibitors specific to particular types of PTP such as NSC 95397, PTP inhibitor IV, CD45 inhibitor, PTP inhibitor II and bpV (bipy). <br />However, there were no statistically significant effects of either one on the maxi-anion channel activation. He could not test all types of PTP due to limited availability of the selective inhibitors. He adopted molecular biological approaches in order to identify the maxi-anion channel-specific PTP. He supposed that receptor tyrosine phosphatases (RPTP) could be better candidates, because they are membrane proteins and would be likely retained in the membrane patches after excision. RPTP&zeta; is one of such phosphatases that are known to be involved in membrane protein dephosphorylation. The mRNA for RPTP&zeta; protein was found in both C127 cells and in cells isolated from the mouse adult fibroblasts (MAF) by the RT-PCR analysis. To test an involvement of RPTP&zeta; in the maxi-anion channel activation, He prepared primary cultures of MAFs from wild-type (WT) and RPTP&zeta;-knockout (RPTP&zeta;-KO) mice. Membrane patches derived from WT MAFs responded to patch excision with robust activation of maxi-anion channels, which showed properties similar to those observed in C127 cells. When He examined the excision-induced maxi-anion channel activation, He found that the channel activation rate was significantly slower in RPTP&zeta;-KO MAFs compared to MAFs from WT mice. Transfection of wild-type, but not the dominant-negative (dn) mutant, RPTP&zeta; into RPTP&zeta;-KO MAFs recovered the maxi-anion channel activation rate to the level comparable to that of the WT MAFs. These results strongly suggest that RPTP&zeta; represents an important part of the excision-induced activation mechanism of the maxi-anion channel. It should be noted, however, that the maxi-anion channel current was not completely eliminated in RPTP&zeta;-KO MAFs. He supposes that some other type(s) of phosphatase could also be involved in dephosphorylation of the maxi-anion channel protein upon membrane patch excision.<br />　　　Next, He used cell-attached mode of patch-clamp in order to retain the integrity of the cells.  Cell swelling in response to hypotonic stimulation is known to activate maxi-anion channels. Indeed, in cell-attached experiment, the activation of the maxi-anion channel occurred after approximately 15 min of application of the hypotonic solution. He examined the effect of vanadate on the hypotonicity-induced maxi-anion channel activation. After 10-min pretreatment with vanadate, the hypotonicity-induced maxi-anion channel activation was significantly suppressed. This result suggests that tyrosine  dephosphorylation play a role in hypotonicity-induced activation of the maxi-anion channel in intact cells. <br />　　　The maxi-anion channel has been shown to represent a major ATP-conductive pathway mediating osmotic swelling-induced release of ATP from C127 cells (Sabirov et al. 2001 J. Gen.  Physiol. 118, 251-266). Thus, He expected that some of the drugs above tested would affect swelling-induced ATP release in a way consistent with their modulatory effects on activation of the maxi-anion channel found in isolated membrane patches. Broad-spectrum PTP inhibitors, dephostatin (0.1 mM) and <i>p</i>-bromotetramisol (0.1 mM) effectively suppressed hypotonicity-induced ATP release from C127 cells. These results are consistent with inhibitory effects of these drugs on the maxi-anion channel activation observed in the present patch-clamp experiments. However, orthovanadate had no effect on the ATP release when used at 1 mM, and even enhanced hypotonicity-induced ATP release at higher concentrations. He interprets this result by effects of this non-specific drug on some other processes involved in hypotonicity-induced ATP release, possibly exocytosis or some other pathways. Consistent with the present patch-clamp data, a broad-spectrum Ser/Thr kinase inhibitor, H7, had no significant effect on swelling-induced release of ATP. In contrast, a tyrosine kinase  inhibitor, genistein, but not its inactive analog, daidzein, had a stimulatory effect on ATP release upon hypotonic stimulation. These results parallel the enhancing effect of genistein on the maxi-anion channel activation in excised inside-out patches. Another non-specific tyrosine kinase inhibitor, AG18, had no significant effect on swelling-induced ATP release being in contrast to its enhancing effect of the maxi-anion channel activation. He suppose that the activating effect of this drug on the maxi-anion channel seen in the  experiments with excised inside-out patches was masked by its action on some other systems involved in swelling-induced ATP release from intact C127 cells. Next He compared swelling-induced ATP release between WT and RPTP&zeta;-KO MAFs. In these experiments, time-dependent ATP release from swollen MAFs derived from RPTP&zeta;-KO mice was significantly lower compared to that from WT. This  result suggests that protein phosphatase RPTP&zeta; is involve in the swelling-induced activation of maxi-anion channels or swelling-induced ATP release.  <br />　　　In the present study, He also provided firm evidence for divalent cation-dependent activation of the maxi-anion channel. Although maxi-anion channels can be activated even in the absence of divalent cations, an increase in the concentrations of free Ca<sup>2＋</sup> or Mg<sup>2＋</sup> greatly accelerated the excision-induced maxi-anion channel activation in a time- and concentration-dependent manner. The mechanism of Ca<sup>2＋</sup> effect on the activation of the maxi-anion channel may involve a Ca<sup>2＋</sup>-dependent protein phosphatase. <br /> Therefore, He next tested the effect of high intracellular free Ca<sup>2＋</sup> on the maxi-anion channel activation in the presence of 1 mM MgATP. Under these conditions, 1 μM free [Ca<sup>2＋</sup>] was able to overcome the inhibitory effect of MgATP on the maxi-anion channel. The rescuing effect of high Ca<sup>2＋</sup>-induced channel activation was significantly suppressed by exposure to 1 mM vanadate. In contrast, the calcineurin inhibitor, compound FK520 did not significantly alter the Ca<sup>2＋</sup>-induced activation of the maxi-anion channel. Since RPTP&zeta; activity is Ca<sup>2＋</sup>-independent (T. Shintani and M. Noda: personal communication), our results may suggest an involvement of a Ca<sup>2＋</sup>-dependent tyrosine phosphatase other than RPTP&zeta; in the Ca<sup>2＋</sup>-dependent activation of the channel. <br />　　　Albeit important general biological functions, the molecular entity of the maxi-anion channel has not yet been identified. Recently, Suzuki and Mizuno (Suzuki and Mizuno, 2004. J. Biol. Chem.  279, 22461-22468) have reported that a gene tweety found in Drosophila flightless locus has a structure similar to those of known channels.  The human homologs of tweety (hTTYH1-3) have been suggested to provide the product, which represents a novel large-conductance Ca<sup>2＋</sup>-activated chloride channel,  while a related gene hTTYH1 gave rise to functional expression of the swelling-activated chloride channel. It has been hypothesized that hTTYH1 might be the large-conductance Ca<sup>2＋</sup>-activated chloride channel  (Suzuki, 2006. Exp. Physiol. 91, 141-147). In order to test this attractive hypothesis, He first searched the cells completely lacking the maxi-anion channel activity. Among several cell lines tested, only the HEK293T cell line was found to exhibit no activity of maxi-anion channels when tested in conditions favoring the channel opening in the excised inside-out mode. Next, He transfected two splice variants of the TTYH1 clone (TTYH1-E and TTYH1-SV) into HEK293T cells and assayed the maxi-anion channel activity on 1-5 days after transfection. The TTYH1-E-transfected cells never showed time-dependent rise of membrane conductance up to about 20 min of patch excision when tested by applying +25 mV test pulses. However, patch currents abruptly exhibited somewhat noisy behavior with not well-defined single-channel amplitudes, over 20 min after patch excision. Very  similar results were obtained in TTYH1-SV-transfected cells as well. Moreover, WT HEK293T cells also failed to respond to patch excision with activation of distinct single-channel events, although seal breakdown-like noisy currents often appeared over 20 min after excision.  Importantly, any typical maxi-anion channel activity with a standard amplitude of ~10 pA at +25mV and its time-dependent inactivation at higher positive and negative voltages could never be observed in either nonetrasfected  HEK293T cells or those transfected with TTYH1-E or TTYH1-SV clones. The data did not positively support an idea that the two clones tested alone represent the maxi-anion channel of the phenotype observed in C127 cells.","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第1106号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"生命科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"20 生理科学専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"2007"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"TOYCHIEV, Abduqodir Hodjiakbarovich","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲1106_要旨.pdf","filesize":[{"value":"535.2 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨","url":"https://ir.soken.ac.jp/record/1191/files/甲1106_要旨.pdf"},"version_id":"5d0d9f85-5109-4b93-948d-d4bc73cc8081"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Activation mechanism of the ATP-conductive maxi-anion channel","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Activation mechanism of the ATP-conductive maxi-anion channel"},{"subitem_title":"Activation mechanism of the ATP-conductive maxi-anion channel","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["22"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"1191","relation_version_is_last":true,"title":["Activation mechanism of the ATP-conductive maxi-anion channel"],"weko_creator_id":"1","weko_shared_id":1},"updated":"2023-06-20T15:55:41.424432+00:00"}