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中的动作电位，并控制许多细胞类型的放电频率。K+通道显示出巨大的功能多样性，由于微妙的分子差异和生理调节剂的差异反应。我们研究的总体目标是了解离子通道渗透和门控特性的生理和分子机制。在广泛分布的Kv2.1钾通道中，发现了一种以前未描述的机制，该机制是通道渗透和门控功能的K+依赖性调节的基础。该机制涉及孔外前庭的构象变化，由K+浓度的生理相关变化控制，并显著影响宏观电流幅度、激活速率、失活速率以及内部和外部通道药理学。这些变化在细胞内通道阻滞剂存在下被放大，所述细胞内通道阻滞剂包括临床上使用的III类抗癫痫药和局部麻醉剂。初步数据表明，这种相同的构象变化也是一个重要的心脏K+通道（Kv1.5）中K+和pH依赖性电流调制的基础，该通道是抗心律失常的靶点。我们将使用膜片钳电生理技术，结合分子诱变技术，以了解钾离子和这种钾离子依赖性的通道构象变化，调节通道功能的机制。具体目标之一是研究控制外前庭形态K+依赖性变化的因素。具体目标二将研究K+依赖性构象变化调节通道门控的机制。这些实验将测试几个假设的机制，连接通道孔的门控过程。具体目标三将测试这一假设，即相同的机制是Kv1.5通道的pH和K+依赖性调节的基础，以及更一般的假设，即这种构象变化代表K+通道用于调节电流幅度和门控特性的一般机制。这些实验将有助于了解这种新机制如何调节通道特性。此外，这些实验将导致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，从而在大脑和心脏中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.