MODULATION OF K+ CHANNEL FUNCTION BY PERMEANTIONS

延迟整流钾(K)通道负责塑造所有可兴奋细胞的动作电位，并控制许多类型细胞的放电频率。由于细微的分子差异和对生理调节剂的不同反应，K通道显示出巨大的功能多样性。我们研究的总体目标是了解离子通道渗透和门控特性背后的生理和分子机制。在广泛分布的Kv2.1钾通道中，发现了一种先前未描述的机制，该机制依赖于钾对通道的渗透和门控功能的调节。这一机制涉及毛孔外前庭的构象变化，受生理上与钾浓度相关的变化控制，并显著影响宏观电流幅度、激活率、失活率和内外通道药理。这些变化在细胞内通道阻滞剂的存在下被放大，这些阻滞剂包括临床使用的III类抗心律失常药物和局部麻醉药。初步数据表明，同样的构象变化也是重要的心脏K通道(Kv1.5)电流依赖于K和pH的调制的基础，Kv1.5是抗心律失常的靶点。我们将使用膜片钳电生理学技术，结合分子突变技术，来了解钾以及这种钾依赖的通道构象变化，调节通道功能的机制。具体目的一是研究控制外前庭钾依赖改变的因素。具体目标二将研究依赖于K的构象变化调制通道门控的机制。这些实验将测试几个关于将通道孔与门控过程联系起来的机制的假设。具体目标三将检验这样的假设，即同样的机制是Kv1.5通道依赖于pH和K的调制的基础，以及更一般的假设，即这种构象变化代表了K通道用来调制电流幅度和门控特性的一般机制。这些实验将导致对这一新机制如何调制信道特性的理解。此外，这些实验将有助于更好地理解细胞内通道阻滞剂如何与外部pH和K相互作用，从而在大脑和心脏产生生理和病理后果。

Delayed rectifier potassium (K+) channels are responsible for shaping the action potential in all excitable cells, and control firing frequency in many cell types. K+ channels display an enormous range of functional diversity, due to subtle molecular differences and differential responsiveness to physiological modulators. The overall goal of our research is to understand the physiological and molecular mechanisms that underlie ion channel permeation and gating characteristics. In the widely distributed Kv2.1 potassium channel, a previously undescribed mechanism was discovered that underlies K+-dependent modulation of both permeation and gating functions of the channel. This mechanism, which involves a conformational change in the outer vestibule of the pore, is controlled by physiologically relevant changes in K+ concentration, and dramatically influences macroscopic current amplitude, activation rate, inactivation rate, and internal and external channel pharmacology. These changes are amplified in the presence of intracellular channel blockers, which include clinically used class III antiarrhythmics and local anesthetics. Preliminary data suggest that this same conformational change also underlies both K+- and pH-dependent modulation of currents in an important cardiac K+ channel (Kv1.5), which is a target for antiarrhythmics. We will use the patch clamp electrophysioloy technique, combined with molecular mutagenesis techniques, to understand the mechanisms by which K , and this K+- dependent change in channel conformation, modulate channel function. Specific aim one will examine the factors that control the K+-dependent change in outer vestibule conformation. Specific aim two will examine the mechanisms by which the K+-dependent conformational change modulates channel gating. These experiments will test several hypotheses regarding the mechanisms that link the channel pore to the gating process. Specific aim three will test the hypothesis that this same mechanism underlies the pH- and K+-dependent modulation of the Kv1.5 channel, and the more general hypothesis that this conformational change represents a general mechanism used by K+ channels to modulate current amplitude and gating properties. These experiments will lead to an understanding of how this novel mechanism modulates channel properties. Furthermore, these experiments will lead to a better understanding of how intracellular channel blockers interact with external pH and K+ to produce physiological and pathological consequences in both brain and heart.