Macías A, de la Cruz A, Prieto A, Peraza D A, Tamkun M M, González T, Valenzuela C
Modelos Experimentales Enfermedades Humanas, Instituto de Investigaciones Biomédicas 'Alberto Sols' (CSIC-UAM), Madrid, Spain.
Br J Pharmacol. 2014 Nov;171(21):4914-26. doi: 10.1111/bph.12822. Epub 2014 Sep 5.
The Kv β1.3 subunit modifies the gating and pharmacology of Kv 1.5 channels in a PKC-dependent manner, decreasing channel sensitivity to bupivacaine- and quinidine-mediated blockade. Cardiac Kv 1.5 channels associate with receptor for activated C kinase 1 (RACK1), the Kv β1.3 subunit and different PKC isoforms, resulting in the formation of a functional channelosome. The aim of the present study was to investigate the effects of PKC inhibition on bupivacaine and quinidine block of Kv 1.5 + Kv β1.3 channels.
HEK293 cells were transfected with Kv 1.5 + Kv β1.3 channels, and currents were recorded using the whole-cell configuration of the patch-clamp technique. PKC inhibition was achieved by incubating the cells with either calphostin C or bisindolylmaleimide II and the effects of bupivacaine and quinidine were analysed.
The voltage-dependent inactivation of Kv 1.5 + Kv β1.3 channels and their pharmacological behaviour after PKC inhibition with calphostin C were similar to those displayed by Kv 1.5 channels alone. Indeed, the IC50 values for bupivacaine were similar in cells whose PKC was inhibited with calphostin C or bisindolylmaleimide II. Similar results were also observed in the presence of quinidine.
The finding that the voltage-dependence of inactivation and the pharmacology of Kv 1.5 + Kv β1.3 channels after PKC inhibition resembled that observed in Kv 1.5 channels suggests that both processes are dependent on PKC-mediated phosphorylation. These results may have clinical relevance in diseases that are characterized by alterations in kinase activity.
Kvβ1.3亚基以蛋白激酶C（PKC）依赖的方式修饰Kv1.5通道的门控和药理学特性，降低通道对布比卡因和奎尼丁介导的阻滞的敏感性。心脏Kv1.5通道与活化C激酶1受体（RACK1）、Kvβ1.3亚基及不同的PKC同工型相关联，从而形成功能性通道体。本研究的目的是探讨PKC抑制对布比卡因和奎尼丁阻断Kv1.5 + Kvβ1.3通道的影响。
将Kv1.5 + Kvβ1.3通道转染至人胚肾293（HEK293）细胞，采用膜片钳技术的全细胞模式记录电流。通过用钙调蛋白C或双吲哚马来酰亚胺II孵育细胞来实现PKC抑制，并分析布比卡因和奎尼丁的作用。
在用钙调蛋白C抑制PKC后，Kv1.5 + Kvβ1.3通道的电压依赖性失活及其药理学行为与单独的Kv1.5通道相似。实际上，在用钙调蛋白C或双吲哚马来酰亚胺II抑制PKC的细胞中，布比卡因的半数抑制浓度（IC50）值相似。在存在奎尼丁的情况下也观察到类似结果。
PKC抑制后Kv1.5 + Kvβ1.3通道的失活电压依赖性和药理学与Kv1.5通道中观察到的相似，这一发现表明这两个过程均依赖于PKC介导的磷酸化。这些结果可能在以激酶活性改变为特征的疾病中具有临床意义。
