Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO 65211, USA.
J Physiol. 2013 Mar 1;591(5):1277-93. doi: 10.1113/jphysiol.2012.241562. Epub 2013 Jan 7.
Abstract β1-Subunits enhance the gating properties of large-conductance Ca(2+)-activated K(+) channels (BKCa) formed by α-subunits. In arterial vascular smooth muscle cells (VSMCs), β1-subunits are vital in coupling SR-generated Ca(2+) sparks to BKCa activation, affecting contractility and blood pressure. Studies in cremaster and cerebral VSMCs show heterogeneity of BKCa activity due to apparent differences in the functional β1-subunit:α-subunit ratio. To define these differences, studies were conducted at the single-channel level while siRNA was used to manipulate specific subunit expression. β1 modulation of the α-subunit Ca(2+) sensitivity was studied using patch-clamp techniques. BKCa channel normalized open probability (NPo) versus membrane potential (Vm) curves were more left-shifted in cerebral versus cremaster VSMCs as cytoplasmic Ca(2+) was raised from 0.5 to 100 μm. Calculated V1/2 values of channel activation decreased from 72.0 ± 6.1 at 0.5 μm Ca(2+)i to -89 ± 9 mV at 100 μm Ca(2+)i in cerebral compared with 101 ± 10 to -63 ± 7 mV in cremaster VSMCs. Cremaster BKCa channels thus demonstrated an ∼2.5-fold weaker apparent Ca(2+) sensitivity such that at a value of Vm of -30 mV, a mean value of [Ca(2+)]i of 39 μm was required to open half of the channels in cremaster versus 16 μm [Ca(2+)]i in cerebral VSMCs. Further, shortened mean open and longer mean closed times were evident in BKCa channel events from cremaster VSMCs at either -30 or 30 mV at any given [Ca(2+)]. β1-Subunit-directed siRNA decreased both the apparent Ca(2+) sensitivity of BKCa in cerebral VSMCs and the appearance of spontaneous transient outward currents. The data are consistent with a higher ratio of β1-subunit:α-subunit of BKCa channels in cerebral compared with cremaster VSMCs. Functionally, this leads both to higher Ca(2+) sensitivity and NPo for BKCa channels in the cerebral vasculature relative to that of skeletal muscle.
摘要 β1 亚基增强由 α 亚基组成的大电导钙激活钾 (BKCa) 通道的门控特性。在动脉血管平滑肌细胞 (VSMCs) 中，β1 亚基对于将 SR 产生的 Ca2+火花耦联到 BKCa 激活至关重要，从而影响收缩性和血压。在睾提肌和脑 VSMCs 中的研究表明，由于功能上的 β1 亚基：α 亚基比值明显不同，因此 BKCa 活性存在异质性。为了定义这些差异，在单个通道水平进行了研究，同时使用 siRNA 来操纵特定的亚基表达。使用膜片钳技术研究了 β1 对 α 亚基 Ca2+敏感性的调节。当细胞质 Ca2+从 0.5 到 100 μm 升高时，与睾提肌 VSMCs 相比，脑 VSMCs 中 BKCa 通道的归一化开放概率 (NPo) 与膜电位 (Vm) 曲线向左移动更多。通道激活的计算 V1/2 值从 0.5 μm Ca2+i 时的 72.0 ± 6.1 mV 降低到 100 μm Ca2+i 时的-89 ± 9 mV，而在睾提肌 VSMCs 中从 101 ± 10 mV 降低到-63 ± 7 mV。因此，睾提肌 BKCa 通道表现出约 2.5 倍的弱的明显 Ca2+敏感性，即当 Vm 值为-30 mV 时，需要平均约 39 μm 的 [Ca2+]i 来打开睾提肌中一半的通道，而在脑 VSMCs 中仅需 16 μm [Ca2+]i。此外，在任何给定的 [Ca2+]下，在-30 或 30 mV 时，从睾提肌 VSMCs 中的 BKCa 通道事件中可以明显观察到缩短的平均开放时间和延长的平均关闭时间。β1 亚基定向 siRNA 降低了脑 VSMCs 中 BKCa 的表观 Ca2+敏感性和自发瞬态外向电流的出现。数据与脑 VSMCs 中 BKCa 通道的 β1 亚基：α 亚基比值高于睾提肌 VSMCs 一致。从功能上讲，这导致脑血管系统中 BKCa 通道的 Ca2+敏感性和 NPo 均高于骨骼肌。
