Lorca Ramón A, Ma Xiaofeng, England Sarah K
Center for Reproductive Health Sciences, Department of Obstetrics and Gynecology, Washington University in St. Louis, St. Louis, Missouri, United States of America.
PLoS One. 2017 Jul 27;12(7):e0182068. doi: 10.1371/journal.pone.0182068. eCollection 2017.
Large conductance voltage- and Ca2+-activated K+ (BKCa) channels are essential regulators of membrane excitability in a wide variety of cells and tissues. An important mechanism of modulation of BKCa channel activity is its association with auxiliary subunits. In smooth muscle cells, the most predominant regulatory subunit of BKCa channels is the β1-subunit. We have previously described that BKCa channels with distinctive N-terminal ends (starting with the amino acid sequence MDAL, MSSN or MANG) are differentially modulated by the β1-subunit, but not by the β2. Here we extended our studies to understand how the distinct N-terminal regions differentially modulate channel activity by β-subunits. We recorded inside-out single-channel currents from HEK293T cells co-expressing the BKCa containing three N-terminal sequences with two β1-β2 chimeric constructs containing the extracellular loop of β1 or β2, and the transmembrane and cytoplasmic domains of β2 or β1, respectively. Both β chimeric constructs induced leftward shifts of voltage-activation curves of channels starting with MANG and MDAL, in the presence of 10 or 100 μM intracellular Ca2+. However, MSSN showed no shift of the voltage-activation, at the same Ca2+ concentrations. The presence of the extracellular loop of β1 in the chimera resembled results seen with the full β1 subunit, suggesting that the extracellular region of β1 might be responsible for the lack of modulation observed in MSSN. We further studied a poly-serine stretch present in the N-terminal region of MSSN and observed that the voltage-activation curves of BKCa channels either containing or lacking this poly-serine stretch were leftward shifted by β1-subunit in a similar way. Overall, our results provide further insights into the mechanism of modulation of the different N-terminal regions of the BKCa channel by β-subunits and highlight the extension of this region of the channel as a form of modulation of channel activity.
大电导电压和Ca2+激活的K+（BKCa）通道是多种细胞和组织中膜兴奋性的重要调节因子。BKCa通道活性调节的一个重要机制是其与辅助亚基的结合。在平滑肌细胞中，BKCa通道最主要的调节亚基是β1亚基。我们之前曾描述过，具有独特N端（起始氨基酸序列为MDAL、MSSN或MANG）的BKCa通道受到β1亚基的差异调节，但不受β2亚基调节。在此，我们扩展了研究，以了解不同的N端区域如何通过β亚基差异调节通道活性。我们记录了共表达包含三种N端序列的BKCa通道与两种β1-β2嵌合构建体的HEK293T细胞的内面向外单通道电流，这两种嵌合构建体分别包含β1或β2的细胞外环以及β2或β1的跨膜和胞质结构域。在存在10或100μM细胞内Ca2+的情况下，两种β嵌合构建体均导致以MANG和MDAL起始的通道电压激活曲线向左移动。然而，在相同的Ca2+浓度下，MSSN的电压激活没有移动。嵌合体中β1的细胞外环的存在类似于完整β1亚基的结果，表明β1的细胞外区域可能是MSSN中观察到缺乏调节的原因。我们进一步研究了MSSN N端区域中存在的一段多聚丝氨酸，观察到含有或缺乏这段多聚丝氨酸的BKCa通道的电压激活曲线都以类似方式被β1亚基向左移动。总体而言，我们的结果为β亚基对BKCa通道不同N端区域的调节机制提供了进一步的见解，并突出了通道该区域的延伸作为通道活性调节的一种形式。
