Functional analysis of plasma membrane Ca2+ATPase 3 and its primary aldosteronism-associated mutations
Functional analysis of plasma membrane Ca2+ATPase 3 and its primary aldosteronism-associated mutations
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质膜Ca2+ATP酶3及其原发性醛固酮增多症相关突变的功能分析
DOI：
10.1016/j.bpj.2022.11.2804
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发表时间：
2023
影响因子：
3.4
通讯作者：
Artigas, Pablo
中科院分区：
文献类型：
--
作者：
Young, Victoria C.;de Sautu, Marilina;Mangialavori, Irene C.;Artigas, Pablo
原发性醛固酮增多症（PA）是继发性高血压最常见的形式，可导致比原发性高血压更高的心血管并发症和死亡率。醛固酮生成腺瘤（APAs）是PA最常见的病因。大多数APAs在质膜离子转运蛋白中存在突变，其中0.6%-9%的APAs在质膜ca2 + atp酶3 （PMCA3）中携带突变。我们开发了一种爪蟾卵母细胞表达系统，以测量PMCA3野生型（PMCA3 WT）和APA相关缺失突变体（PMCA3 L425_V426del.）在最小内源污染下的功能。我们测量了37℃时表达PMCA3的卵母细胞制备的质膜中atp酶的活性。在表达PMCA3 WT的卵母细胞制备物中观察到ca2 +依赖性atp酶，ca2 +依赖性用矩形双曲线（k0.5 = 25.6±5.1 μM）很好地描述（来自三种独立膜制备物的三倍）。PMCA3 L425_V426del。表达的卵母细胞缺乏ca2 +依赖性atp酶活性，表明突变体中酶活性的丧失。我们使用双电极电压钳电生理学来评估pmca3变异介导电流的存在。在静息的细胞内[ca2 +]，注射PMCA3 wt的卵母细胞在所有电压下的电流与未注射PMCA3 wt的卵母细胞的电流没有区别。当细胞外125 mM Na+浸泡，PMCA3 L425_V426del。注射后的卵母细胞呈现负膜电位向内电流和正膜电位向外电流，与异常通道样电流的诱导一致。当外部Na+被n -甲基d -葡萄糖胺+取代时，电流向外定向，对细胞外[ca2 +]的变化不敏感。这些结果表明PMCA3 L425_V426del。导致高醛固酮增多症，这是由于伴随ca2 +运输活性的丧失和Na+介导的去极化电流的诱导。目前的实验正在评估atp酶和其他pa相关的PMCA3突变的生理特征。NSF-MCB 2003251资助。
Primary aldosteronism (PA) is the most common form of secondary hypertension and can lead to higher rates of cardiovascular complications and death than essential hypertension. Aldosterone-producing adenomas (APAs) are the most frequent cause of PA. Most APAs have mutations in plasma membrane ion-transport proteins with 0.6%-9% carrying a mutation in the Plasma Membrane Ca 2+ ATPase 3 (PMCA3). We have developed a Xenopus oocyte expression system to measure the function of PMCA3 wildtype (PMCA3 WT) and an APA associated deletion mutant (PMCA3 L425_V426del.) with minimal endogenous contamination. We measured ATPase activity at 37 C in plasma membrane preparations from oocytes expressing PMCA3. Ca 2+-dependent ATPase was observed in preparations from oocytes expressing PMCA3 WT, with a [Ca 2+] dependence well described with a rectangular hyperbola (K 0.5= 25.6±5.1 μM)(triplicates from three independent membrane preparations). In contrast, membrane preparations from PMCA3 L425_V426del. expressing oocytes lacked Ca 2+-dependent ATPase activity, indicating loss of enzymatic activity in the mutant. We used two-electrode voltage clamp electrophysiology to evaluate the presence of PMCA3-variant mediated currents. At resting intracellular [Ca 2+], PMCA3 WT-injected oocytes had currents indistinguishable from those in uninjected oocytes, at all voltages. When bathed by extracellular 125 mM Na+, PMCA3 L425_V426del.-injected oocytes presented inward currents at negative membrane potentials and outward currents at positive ones, consistent with induction of an aberrant channel-like current. The currents were outwardly directed upon substitution of external Na+ with N-methyl D-glucamine+ and were insensitive to changes in extracellular [Ca 2+]. These results indicate that PMCA3 L425_V426del. causes hyperaldosteronism due to the concomitant loss of active Ca 2+ transport and induction of a depolarizing Na+-mediated current. Current experiments are evaluating the ATPase and physiological characteristics of other PA-associated PMCA3 mutations. Funded by NSF-MCB 2003251.