Name: characterizing mammalian zinc transporters using an in vitro zinc transport assay
Uploaded: 2023-06-02T13:20:00.000+00:00
Duration: 7 min 55 s
Description: Watch this Scientific Journal Video about Exploring Cellular Zinc Regulation Through ZnT1 Functionality at JoVE.com

Raz Zarivach 得到了以色列科学基金会的支持（资助号:163/22）。Tomer Eli Ben Yosef 和 Arie Moran 得到了以色列科学基金会的支持（资助号:2047/20）。我们要感谢丹尼尔·吉特勒（Daniel Gitler）及其在本古里安大学（Ben-Gurion University）的团队的合作、支持和专业知识。

1. 细胞转染
<ol><li>在补充有10%胎牛血清（FBS），2mM L-谷氨酰胺和1x青霉素/链霉素（参见 材料表）的Dulbecco改良Eagle培养基（DMEM）中培养HEK293T细胞在37°C / 5%CO2 的湿润培养箱中培养，直到汇合在10cm平板上（8.8×106 个细胞）。</li>
	<li>将一个 13 mm 盖玻片放入 12 孔板的每个孔中。在 12 mL 完全 DMEM 中稀释步骤 1.1 中的 0.44 x10 6 个胰蛋白酶消化细胞。通过上下移液三到五次充分混合。用 1 mL 混合溶液填充每个孔。在37°C / 5%CO2 的潮湿培养箱中生长过夜。</li>
	<li>用每个孔中 1 mL 无血清 DMEM（参见 材料表）替换每个孔中的完整 DMEM。如步骤1.1所示，将12孔板放回加湿的培养箱中。</li>
	<li>对于每个转染孔，在 1.5 mL 试管中的 100 μL 无血清 DMEM 中稀释 1 μg 含有用 mCherry 荧光蛋白标记的目标蛋白（补充文件 1）的 pAAV2 质粒。</li>
	<li>每次转染将 3 μg 聚乙烯亚胺（PEI，参见 材料表）加入 100 μL 无血清 DMEM 中，每次转染在不同的 1.5 mL 试管中。质粒:PEI比例应为1:3（μg:μg）。</li>
	<li>将步骤1.4和步骤1.5中的两个管涡旋10秒，并在室温下静置至少5分钟。</li>
	<li>将步骤1.4中的一份（每孔100μL）DNA溶液与步骤1.5中的一份（每孔100μL）PEI溶液混合。涡旋最终溶液10秒，并在室温下静置至少20分钟，最多6小时。</li>
	<li>从培养箱中取出 12 孔板。涡旋步骤1.7的最终溶液，并向步骤1.3的12孔板中的每个孔中加入200μL。将 12 孔板放回加湿培养箱（步骤 1.1）。</li>
	<li>3 小时后，用 1 mL 完全 DMEM 替换每个孔中的培养基。如步骤1.1所示，将12孔板放回加湿的培养箱中。</li>
	<li>2天后，从37°C / 5%CO2 培养箱中取出转染的细胞培养物，并使用10倍放大倍率将其置于倒置荧光显微镜上。</li>
	<li>使用显微镜对焦轮，在使用明场光的同时聚焦在细胞上。</li>
	<li>切换到荧光 mCherry 激发 （587 nm） 和发射 （610 nm） 波长，并关闭明场光。检查细胞的荧光以确认ZnT1 mCherry的表达。</li>
	<li>准备染料上样液。<ol><li>从储存在-20°C冰箱中的避光原液中取出4μL锌特异性荧光染料（参见 材料表）的乙酰氧基甲基（AM）酯形式的等分试样，溶解在DMSO（1μg/ μL）中。这种形式允许染料通过简单的扩散通过质膜进入细胞。</li>
		<li>将 4 μL 10% 普鲁酸（或 2 μL 20% 普鲁酸，参见 材料表）加入步骤 1.13.1 的等分试样中。通过上下移液 3 次充分混合溶液，然后将所有 8 μL 加入 1.5 mL 试管中。</li>
		<li>从步骤1.13.2开始，向试管中加入补充有1mg / mL（~0.1%）牛血清白蛋白的750μL林格氏溶液（内部制备，参见 成分表 ），并剧烈涡旋。再加入 750 μL 相同的溶液，再次涡旋以确保最大混合。</li>
	</ol></li>
	<li>将步骤 1.13.3 中的 750 μL 最终溶液加入新的 6 孔培养板中的两个孔中。用铝箔盖住。 注意:为避免漂白，从此步骤开始，6孔板始终用铝箔覆盖。</li>
	<li>使用细镊子，从转染的细胞培养板中取出最多四个重复的盖玻片，并将它们置于步骤1.14中新的6孔板的第一个填充孔中（每孔最多四个载玻片）。对第二个填充孔重复此过程。 注意:同一填充孔中的所有盖玻片必须来自相同的条件。但是，每个井可以包含不同的条件。</li>
	<li>盖上铝箔，轻轻摇晃15-20分钟。</li>
	<li>除去染料上样液，并用新的林格氏加白蛋白洗涤液代替，如步骤1.13.3所示。用铝箔盖住。再次摇晃 20 分钟。这允许细胞内酯酶裂解AM酯。</li>
</ol>2. 显微镜准备
<ol><li>如前所述安排必要的工具（参见 材料表）:能够检测 GFP 和 mCherry 荧光团的倒置荧光显微镜、允许在至少两种溶液之间切换的灌注系统、抽吸系统和灌注室。</li>
	<li>打开显微镜、显微镜的光源和相机。打开抽吸系统。</li>
	<li>清洗灌注系统。用林格氏溶液洗涤第一个腔室，用含有7μM锌溶液的林格氏溶液洗涤第二个腔室，该溶液补充有7μM吡啶硫酮（锌离子载体，参见 材料表）。为避免气泡或间隙，请在每个腔室中留出一些液体。 注意:由于两个容器都连接到连接到灌注室的同一根管子上，因此请始终确保用林格溶液洗涤共享段。</li>
	<li>关闭水龙头，并用林格溶液和林格锌溶液填充适当的腔室，如步骤2.3所示。</li>
</ol>3. 样品制备
<ol><li>取灌注室，将其放置，凹槽的窄边朝上。 注意:凹槽是灌注室中间的一个孔。它允许灌注液进入细胞。凹槽的一侧较窄，另一侧较宽。</li>
	<li>在凹槽周围涂上密封硅胶（参见 材料表）。使用移液器吸头清洁凹槽中的任何密封硅胶。 注意: 确保凹槽周围有足够的硅胶密封件来密封 22 毫米的盖玻片。</li>
	<li>使用细镊子，从洗涤液中取出盖玻片，并将其放在凹槽顶部，细胞朝下。这样，细胞将在实验过程中暴露于灌注凹槽的溶液中。</li>
	<li>将 22 毫米盖玻片放在 13 毫米盖玻片的顶部，然后使用镊子拧紧。注意不要使盖玻片破裂。</li>
	<li>翻转腔室，然后按压它以释放所有存在的液体。用 100 μL 林格氏洗涤液填充凹槽，然后再次按压以确保无泄漏。 注意: 如果检测到泄漏，请在泄漏部位涂抹密封剂，然后重新测试。</li>
	<li>将灌注室安装到平台上，并将其固定。放置灌注管和抽吸管，使灌注在凹槽中的细胞上。</li>
	<li>将灌注速率更改为约 2 mL/min，然后打开林格氏溶液的灌注。确保灌注系统在没有泄漏或溢出的情况下工作。</li>
</ol>4. 测量准备
<ol><li>双击图标打开成像软件（参见 材料表）。使用相关凭据登录。选择连接的相机，然后按 确定。</li>
	<li>使用显微镜左侧的按钮将显微镜放大倍率设置为10倍。</li>
	<li>选择实时取景并使用操纵杆，移动平台以聚焦于凹槽中的单元格。</li>
	<li>灌注打开时，关闭灯，按下界面中的专用按钮将波长更改为 mCherry。使用显微镜对焦轮调整焦距。</li>
	<li>显微镜聚焦在细胞上后，使用操纵杆移动平台以选择合适的细胞贴片。 注意:合适的区域被认为是具有至少 10 个单元格的区域（用于 ROI）和空白区域（用于背景）。</li>
	<li>选择打开/关闭 ROI 下拉菜单，然后选择绘制圆形 ROI。</li>
	<li>用表达mCherry的细胞绘制细胞簇的ROI（指示ZnT1的表达）。</li>
	<li>在与步骤 4.6 相同的工具栏中，单击" 打开/关闭背景 ROI" 按钮，然后将背景 ROI 的位置和大小调整为完全没有单元格的区域。<ol><li>检查 mCherry 和 EGFP 波长，以确保所选背景 ROI 中没有细胞。 注意:调整波长，使mCherry使用520 nm的激发波长和610 nm的发射波长，EGFP使用470 nm的激发波长和520 nm的发射波长。</li>
	</ol></li>
	<li>选择 波长 （λ） 子选项卡。标记其复选框，并确保存在 GFP 波长并标记其复选框。如果没有，请添加并标记它。</li>
	<li>选择 "持续时间 "子选项卡。将测量间隔定义为每 5 秒一次，并更改间隔数以适应实验持续时间。</li>
	<li>在目镜下方显微镜面板上的"对焦"部分，单击"打开"按钮以启用完美对焦系统 （PFS）。</li>
	<li>使用 PFS 对焦轮调整对焦。在软件界面中，在 ND 采集下，确保勾选 PFS on 。</li>
	<li>单击" 立即运行"。</li>
</ol>5.实验步骤
<ol><li>首先使用 Ringer 溶液灌注进行 90 秒基线期测量。</li>
	<li>基线期结束后，关闭林格氏溶液灌注，然后打开林格氏锌溶液灌注。通过单击主界面面板右下角的红旗来标记解决方案的切换。预计会出现荧光的上升。</li>
	<li>一旦荧光上升开始饱和，就用林格氏溶液变回灌注。关闭林格氏锌溶液灌注，然后打开林格氏溶液灌注。</li>
	<li>等到实验时间到期。如果 ZnT1 工作正常，预计荧光会明显而稳定地下降。</li>
</ol>6. 数据导出
<ol><li>要导出带有基线减法的数据，请按减 去基线 按钮，然后在"ND采集窗口"中查看更改。</li>
	<li>单击"ND 采集窗口"软件界面中的 "导出 "按钮。将打开一个 excel 数据表。保存到所需位置。</li>
</ol>7. 数据分析
<ol><li>对于每个 ROI，从前 90-100 秒创建平均基线荧光。</li>
	<li>将为每个 ROI 计算的荧光表示为该 ROI 的背景百分比。</li>
	<li>创建所有 ROI 的行平均值，并将结果绘制为折线图。这将创建所有 ROI 的平均值作为时间的函数。</li>
	<li>使用线性拟合功能，选择用林格氏溶液洗涤后荧光降低的初始速率。方程的斜率值与传输速率相关。</li>
</ol>

使用 FluoZin-3 测定研究哺乳动物细胞中的锌转运机制

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zinc+indicator+FluoZin-3+is+not+perturbed+significantly+by+physiological+levels+of+calcium+or+magnesium.">The zinc indicator FluoZin-3 is not perturbed significantly by physiological levels of calcium or magnesium.</a> Cell Calcium. 44 (4), 422-426 (2008).</li><li>Gee, K. R., Zhou, Z. -. L., Ton-That, D., Sensi, S. L., Weiss, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+zinc+in+living+cells.:+A+new+generation+of+sensitive+and+selective+fluorescent+probes.">Measuring zinc in living cells.: A new generation of sensitive and selective fluorescent probes.</a> Cell Calcium. 31 (5), 245-251 (2002).</li><li>Sensi, S. L., Ton-That, D., Weiss, J. H., Rothe, A., Gee, K. 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M., Cuttle, M., Mittoo, S., Bradley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsphere-based+real-time+calcium+sensing.">Microsphere-based real-time calcium sensing.</a> Angewandte Chemie International Edition. 45 (33), 5472-5474 (2006).</li><li>Namdarghanbari, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+of+the+zinc+sensor+FluoZin-3+with+Zn7-metallothionein:+Inquiry+into+the+existence+of+a+proposed+weak+binding+site.">Reaction of the zinc sensor FluoZin-3 with Zn7-metallothionein: Inquiry into the existence of a proposed weak binding site.</a> Journal of Inorganic Biochemistry. 104 (3), 224-231 (2010).</li><li>Devinney, M. J., Reynolds, I. J., Dineley, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+of+intracellular+free+calcium+and+zinc+using+fura-2FF+and+FluoZin-3.">Simultaneous detection of intracellular free calcium and zinc using fura-2FF and FluoZin-3.</a> Cell Calcium. 37 (3), 225-232 (2005).</li></ol>

作者声明没有利益冲突。

上述方法允许以高时间分辨率直接测量细胞内锌浓度。与其他方法相比，这种涉及监测细胞内Zn2+变化的方法可以显着降低背景噪声。此外，染料对锌的选择性消除了与其他金属阳离子的潜在交叉相互作用18,19。最后，它没有直接的细胞毒性，可以测试活细胞过程19。
但是，这种方法有一定的局限性。首先?...

锌是细胞环境中必需的微量元素。它包含所有蛋白质的三分之一，并参与各种细胞过程，例如催化1、转录2 和结构基序3。然而，尽管是氧化还原惰性的，但高浓度的锌对细胞有毒，这就是为什么没有哺乳动物生物体在没有调节锌稳态的机制的情况下生存的原因。在哺乳动物中，有三种机制负责这一过程:（1）金属硫蛋白，它是富含胞质半胱氨酸的蛋白质，以高亲和力结合锌，从而防止过量的游离胞质锌4;（2） Zrt/Irrt 样蛋白 （ZIP），它们是锌转运蛋白，负责锌通过质膜或细胞内细胞器流入胞质溶胶 4,5,6,7,8;（3） ZnTs，是无处不在的阳离子扩散促进剂 （CDF） 家族的哺乳动物亚群，是锌转运蛋白，因为它们将锌从胞质溶胶挤出穿过质膜或进入细胞内细胞器4,5,6,7,8,9。由于锌对细胞代谢的重要性，了解细胞锌动力学至关重要。
以前评估锌动力学的方法依赖于通过将mRNA与固定组织或细胞的细胞锌测量值相关联来评估不同锌条件下mRNA的表达水平10,11,12。这些方法包括化学检测和免疫组化染色。然而，这些方法只能产生间接测量，因此只能确定细胞内锌浓度与锌转运蛋白表达之间的离线相关性。因此，这些方法无法推断任何需要高时间分辨率的参数。
锌2+ 输运的更直接测量使用锌13的放射性同位素。该方法依赖于放射性标记Zn2+ 的测量来监测锌的传输及其动力学。然而，由于锌对细胞稳态的重要性，多个细胞过程调节细胞内锌浓度。其中包括细胞外结合和几种协同工作以维持细胞内 Zn2+ 水平的严格控制的运输系统。这些过程的结合会产生相当大的背景噪声，这使得测试单个与锌相关的传递函数变得困难。
本文展示了一种通过使用锌特异性荧光染料 FluoZin-3 测量细胞内游离锌浓度来直接监测锌转运速率的方法。该染料对Zn2+ 具有高度特异性，并且几乎不受其他二价阳离子（如钙）的干扰。此外，以酯的形式，它通过非离子扩散进入细胞，然后由于细胞内二酯酶的活性而被捕获。因此，其荧光主要与游离胞质锌浓度相关。这些实验旨在研究锌转运蛋白1（ZnT1）的结构-功能关系，ZnT1是ZnT家族的成员。

<table><tbody><tr><td>10 cm plate</td><td>greiner bio-one</td><td>664160</td><td></td></tr><tr><td>12-well cell culture plate</td><td>greiner bio-one</td><td>665180</td><td></td></tr><tr><td>13 mm coverslips</td><td>Superior Marienfeld</td><td>111530</td><td></td></tr><tr><td>22 mm cover slides</td><td>Superior Marienfeld</td><td>101050</td><td></td></tr><tr><td>6-well culture plate</td><td>greiner bio-one</td><td>657160</td><td></td></tr><tr><td>Bovine serum albumin</td><td>bioWorld</td><td>22070008</td><td></td></tr><tr><td>Calcium chloride anhydrous, granular</td><td>Sigma Aldrich</td><td>C1016</td><td>Concentration in Ringer solution: 1 mM</td></tr><tr><td>D-(+)-Glucose</td><td>Glentham Life Science</td><td>GC6947</td><td>Concentration in Ringer solution: 10 mM</td></tr><tr><td>Dubelco&amp;rsquo;s Modified Eagle Media (DMEM)&amp;nbsp;</td><td>Sartorius</td><td>01-055-1A</td><td></td></tr><tr><td>Eclipse Ti inverted microscope</td><td>Nikon</td><td>TI-DH</td><td>Discontinued. Replaced by Eclipse Ti2</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Cytiva</td><td>SH30088.03</td><td></td></tr><tr><td>Fine tweezers</td><td>Dumont</td><td>0203-55-PS</td><td></td></tr><tr><td>Fluozin-3AM</td><td>Invitrogen</td><td>F24195</td><td></td></tr><tr><td>HyClone Penicillin-Streptomycin 100x solution</td><td>Cytiva</td><td>SV30010&amp;nbsp;</td><td></td></tr><tr><td>LED illumination system</td><td>CoolLED</td><td>pE-4000</td><td></td></tr><tr><td>L-glutamine</td><td>Biological Industries</td><td>03-020-1B</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Merck</td><td>1.05833</td><td>Concentration in Ringer solution: 0.8 mM</td></tr><tr><td>N[2-Hydroxyethyl]piperazine-N&#039;-[2-ethanesulfonic acid] (HEPES)</td><td>Formedium</td><td>HEPES10</td><td>Concentration in Ringer solution: 10 mM</td></tr><tr><td>Neo 5.5 sCMOS camera</td><td>ANDOR</td><td>DC-152Q-FI</td><td></td></tr><tr><td>NIS-Elements imaging software</td><td>Nikon</td><td>AR</td><td></td></tr><tr><td>Pluronic acid F-127</td><td>Millipore</td><td>540025</td><td></td></tr><tr><td>Pottasium chloride</td><td>Bio-Lab</td><td>163823</td><td>Concentration in Ringer solution: 5.4 mM</td></tr><tr><td>Pyrithione</td><td>Sigma Aldrich</td><td>H3261</td><td>Concentration in Ringer zinc solution: 7 &amp;mu;M</td></tr><tr><td>Silicone Grease Kit</td><td>Warner Instruments</td><td>W4 64-0378</td><td></td></tr><tr><td>Sodium chloride</td><td>Bio-Lab</td><td>190305</td><td>Concentration in Ringer solution: 120 mM</td></tr><tr><td>Zinc sulfate</td><td>Sigma Aldrich</td><td>31665</td><td>Concentration in Ringer zinc solution: 7 &amp;mu;M</td></tr></tbody></table>

characterizing mammalian zinc transporters using an in vitro zinc transport assay

过渡金属（如Zn2+离子）由于其细胞毒性而必须受到严格调控。以前，通过测定不同浓度Zn2+下转运蛋白的表达水平来间接测量Zn2+转运蛋白的活性。这是通过利用免疫组化、测量组织中的 mRNA 或确定细胞 Zn2+ 水平来完成的。随着细胞内Zn 2+传感器的发展，锌转运蛋白的活性目前主要通过关联使用荧光探针检测的细胞内Zn 2+的变化与Zn2+转运蛋白的表达来确定。然而，即使在今天，也只有少数实验室监测细胞内Zn2+的动态变化，并用它来直接测量锌转运蛋白的活性。部分问题在于，在ZnT家族的10种锌转运蛋白中，除了ZnT10（转运锰）外，只有锌转运蛋白1（ZnT1）位于质膜上。因此，将转运活性与细胞内Zn2+浓度的变化联系起来是很困难的。本文介绍了一种使用基于锌特异性荧光染料 FluoZin-3 的测定法确定锌传输动力学的直接方法。这种染料以酯的形式加载到哺乳动物细胞中，然后由于细胞二酯酶活性而被困在胞质溶胶中。细胞利用锌2+ 离子载体吡硫酮加载锌 2+。ZnT1活性从细胞洗脱后荧光减少的线性部分评估。在 470 nm 激发和 520 nm 发射下测量的荧光与游离细胞内 Zn2+ 成正比。选择用mCherry荧光基团标记的表达ZnT1的细胞可以仅监测表达转运蛋白的细胞。该测定用于研究 ZnT1 蛋白的不同结构域对人 ZnT1 转运机制的贡献，ZnT1 是一种从细胞中挤出过量锌的真核跨膜蛋白。

ZnT1 是一种哺乳动物锌转运蛋白，位于细胞质膜上13。它是阳离子扩散促进剂 （CDF） 蛋白家族的成员，可将锌从胞质溶胶挤出到细胞外毫14。ZnT1 具有双结构域结构:跨膜结构域（跨膜结构域）和 C 端结构域14。与其他已知的 CDF 蛋白不同，ZnT1 具有扩展的非结构化 C 末端结构域 （USCTD）。USCTD的作用目前尚不清楚。我们使用上述方案将ZnT1 WT与没?...

Watch this Scientific Journal Video about Exploring Cellular Zinc Regulation Through ZnT1 Functionality at JoVE.com

作者聚焦:通过 ZnT1 功能探索细胞锌调节 

由于与蛋白质功能的因果关系较弱且时间分辨率低，锌转运已被证明具有挑战性。该协议描述了一种通过使用Zn 2 +敏感荧光染料以高时间分辨率监测活细胞中Zn 2 +挤出的方法，从而提供Zn2 +外排的直接测量。

使用 体外 锌转运测定表征哺乳动物锌转运蛋白

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...

主要研究哺乳动物锌转运蛋白的构能关系，特别强调其调控机制。由于锌离子对细胞功能的重要性，因此很难准确测量细胞锌动力学。我们的协议解决了锌动力学的低信噪比和低时间分辨率的问题。该方法允许测试锌流入细胞膜，具有高时间分辨率，并且显着减少背景噪声和其他二价阳离子的交叉相互作用。我们发现哺乳动物转运蛋白 ZnT1 的一个 C 末端结构域的非结构化延伸不参与锌转运功能。这使研究人员能够专注于 ZnT1 蛋白的其他结构域以进行锌转运调节。

characterizing-mammalian-zinc-transporters-using-an-vitro-zinc

Research

JoVE Journal

Biology

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

1.1K 次观看.  Ben-Gurion University. 主要研究哺乳动物锌转运蛋白的构能关系，特别强调其调控机制。由于锌离子对细胞功能的重要性，因此很难准确测量细胞锌动力学。我们的协议解决了锌动力学的低信噪比和低时间分辨率的问题。该方法允许测试锌流入细胞膜，具有高时间分辨率，并且显着减少背景噪声和其他二价阳离子的交叉相互作用。我们发现哺乳动物转运蛋白 ZnT1 的一个 C 末端结构域的非结构?...

视频: 作者聚焦:通过 ZnT1 功能探索细胞锌调节 

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

原子吸纳光谱测量哺乳动物细胞细胞内锌池

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

科研

生物化学

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

内向整流钾通道的小分子调节剂的高通量筛选

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

生物学

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein structure. Metal sequestration by hosts often leads to metal limitation for the bacterium. This limitation induces bacterial gene expression whose protein products allow bacteria to overcome their metal-limited environment. Characterization of such genes is challenging. Bacteria must be grown in meticulously prepared media that allows sufficient access to nutritional metals to permit bacterial growth while maintaining a metal profile conducive to achieving expression of the aforementioned genes. As such, a delicate balance must be established for the concentrations of these metals. Growing a nutritionally fastidious organism such as Neisseria gonorrhoeae, which has evolved to survive only in the human host, adds an additional level of complexity. Here, we describe the preparation of a defined metal-limited medium sufficient to allow gonococcal growth and the desired gene expression. This method allows the investigator to chelate iron and zinc from undesired sources while supplementing the media with defined sources of iron or zinc, whose preparation is also described. Finally, we outline three experiments that utilize this media to help characterize the protein products of metal-regulated gonococcal genes.

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands

We describe here a method for growth of Neisseria gonorrhoeae in metal-restricted liquid medium to facilitate the expression of genes for metal uptake. We also outline downstream experiments to characterize the phenotype of gonococci grown in these conditions. These methods may be adapted to be suitable for characterization of metal-responsive genes in other bacteria.

内塞里亚淋病的金属有限生长，用于金属反应基因特征和从宿主配体中获取金属

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein ...

免疫与感染

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

测量离子转运的Na，K，H，K-ATP酶活性的<em&gt;爪蟾</em卵母细胞的原子吸收分光光度法：另一种放射性同位素检测

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

We present a protocol for the in vitro investigation of efflux pumps from Pseudomonas aeruginosa. This protocol allows for the generation of a robust, reversible, and tunable proton gradient within the liposome membrane and hence should be adaptable to any membrane protein energized by the protomotive force.

在 MexAB外排泵从铜绿假单胞菌的体外研究

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质<sup&gt; -</sup&gt;通道和转运

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

蛋白质直接送货到哺乳动物细胞利用细胞渗透半胱氨酸<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt;锌指域

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a systematic approach combining different methodologies to assess the availability of zinc (Zn) in Atlantic salmon (Salmo salar). Considering that several Zn chemical species can be present in an Atlantic salmon feed, it was hypothesised that Zn availability is influenced by the Zn chemical species present in the feed. Thus, in this study, the first protocol is about how to extract the different Zn chemical species from the feed and to analyze them by a size exclusion chromatography-inductively coupled plasma mass spectroscopy (SEC-ICP-MS) method. Subsequently, an in vitro method was developed to evaluate the solubility of dietary Zn in Atlantic salmon feeds. The third protocol describes the method to study the impact of changing Zn chemical species composition on the uptake of Zn in a fish intestinal epithelial model using a rainbow trout gut cell line (RTgutGC). Together, the findings from the in vitro methods were compared with an in vivo study examining the apparent availability of inorganic and organic sources of Zn supplemented to Atlantic salmon feeds. The results showed that several Zn chemical species can be found in feeds and the efficiency of an organic Zn source depends very much on the amino acid ligand used to chelate Zn. The findings of the in vitro methods had less correlation with that outcome of the in vivo study. Nevertheless, in vitro protocols described in this article provided crucial information regarding Zn availability and its assessment in fish feeds.

This article explains in detail a systematic approach to assess micro-mineral availability in Atlantic salmon. The methodology includes tools and models with increasing biological complexity: (1) chemical speciation analysis, (2) in vitro solubility, (3) uptake studies in cell lines, and (4) in vivo fish studies.&#160;

使用大西洋鲑鱼锌示例证明的补充方法评估鱼类饲料中的矿物供应情况

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a ...

Assessing Bioindicator Phenotypes for Zinc Limitation in M. smegmatis

Using Mycobacterium smegmatis as a Bioindicator for Zinc-Limited Growth Conditions in Mycobacteria

Many bacteria build alternative ribosomes in Zn2+-limiting growth conditions by replacing Zn2+-binding ribosomal proteins with Zn2+-independent paralogs. Defining a system to study these alternative ribosomes has proven difficult because Zn2+ contamination in the laboratory is common. To address this issue, chelating agents are sometimes added to growth media, but this approach convolutes the biological response to gradual Zn2+&#160;limitation and is associated with ribosome hibernation. Here, detailed instructions are outlined for preparing media and seeding cultures for Zn2+-limited growth without adding chelators. Following this method, the model bacterium, Mycobacterium smegmatis, undergoes morphogenesis, which depends on alternative ribosomes. Because morphogenesis is tractable and only occurs in Zn2+-limiting conditions, M. smegmatis can be used as a bioindicator to verify biologically relevant growth conditions. Three bioindicator phenotypes (cell density, cell length, and coenzyme F420 fluorescence) that indicate Zn2+ limitation in the wild-type are described, and changes in these bioindicators for a deletion mutant that cannot build alternative ribosomes are outlined. Since trace Zn2+ contamination is difficult to control for each batch of media, and precise quantification of Zn2+ in each media preparation is overly burdensome, following this bioindicator phenotype is an accessible way to validate the preparation of Zn2+-limited growth media. To help identify proper conditions for Zn2+-limiting growth and alternative ribosome production, changes in the bioindicator phenotypes were profiled for Zn2+-contaminated or severely Zn2+-depleted preparations of Zn2+-limited media as well. Further details to achieve Zn2+-limiting growth and alternative ribosome production in M. tuberculosis are presented, along with the associated bioindicator phenotype. Overall, the detailed instructions and bioindicator phenotypes described here will help standardize the production of translationally active alternative ribosomes in mycobacteria.

This protocol details preparation of Zn2+-limited media, growth vessels, and seed cultures to produce Zn2+-limited ribosomes in mycobacteria. Traces of Zn2+ lead to Zn2+-limiting growth and we describe morphogenic features used as bioindicators to verify Zn2+ limitation in vitro in the model bacterium, Mycobacterium smegmatis, and the human pathogen M. tuberculosis.

Many bacteria build alternative ribosomes in Zn2+-limiting growth conditions by replacing Zn2+-binding ribosomal proteins with Zn2+-independent paralogs. ...

使用体外锌转运测定表征哺乳动物锌转运蛋白

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此视频中的章节

原子吸纳光谱测量哺乳动物细胞细胞内锌池

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

内向整流钾通道的小分子调节剂的高通量筛选

内塞里亚淋病的金属有限生长，用于金属反应基因特征和从宿主配体中获取金属

测量离子转运的Na，K，H，K-ATP酶活性的

在 MexAB外排泵从铜绿假单胞菌的体外研究

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质

蛋白质直接送货到哺乳动物细胞利用细胞渗透半胱氨酸

使用大西洋鲑鱼锌示例证明的补充方法评估鱼类饲料中的矿物供应情况

Using Mycobacterium smegmatis as a Bioindicator for Zinc-Limited Growth Conditions in Mycobacteria

使用 体外 锌转运测定表征哺乳动物锌转运蛋白

副本

摘要

探索更多视频

此视频中的章节

原子吸纳光谱测量哺乳动物细胞细胞内锌池

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

内向整流钾通道的小分子调节剂的高通量筛选

内塞里亚淋病的金属有限生长，用于金属反应基因特征和从宿主配体中获取金属

测量离子转运的Na，K，H，K-ATP酶活性的

在 MexAB外排泵从铜绿假单胞菌的体外研究

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质

蛋白质直接送货到哺乳动物细胞利用细胞渗透半胱氨酸

使用大西洋鲑鱼锌示例证明的补充方法评估鱼类饲料中的矿物供应情况

Using Mycobacterium smegmatis as a Bioindicator for Zinc-Limited Growth Conditions in Mycobacteria

使用体外锌转运测定表征哺乳动物锌转运蛋白