Stephen J. Korn - US grants

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.

DESCRIPTION (provided by applicant): Voltage-activated potassium channels serve several critical functions in all excitable cells (brain, heart, endocrine and muscle), including repolarization of action potentials and control of rhythmic firing patterns. Channel properties that control the functional outcome of channel activity include current magnitude and the rate of channel gating events (activation, inactivation and deactivation). The functionally rich outer vestibule/selectivity filter region of the potassium channel pore has been considered to have just a single conformation in the open state, and have little or no role in the modulation of open channel function. Recently, we described a novel mechanism by which current magnitude, activation rate and inactivation rate, as well as both internal and external channel pharmacology, are modulated by relevant changes in external potassium concentration. We demonstrated that changes in these channel properties, which can be substantial, result from a previously unknown type of conformational change that occurs in the outer vestibule of the pore. Furthermore, this conformational change in the outer vestibule is observed only in channels that display properties consistent with a "structurally flexible" selectivity filter region of the pore. These results suggest the possibility that, in contrast to what has been previously believed, the permeation pathway in some ion channels has a significant degree of "structural flexibility," and that this "flexibility" can markedly affect open channel function. Neither the nature of the conformational change, nor a detailed understanding of how it is regulated, can be obtained solely with electrophysiological techniques. The goal of this application is to integrate two sophisticated fluorescence techniques, fluorescence quenching and fluorescence resonance energy transfer (FRET), with patch clamp electrophysiology in our lab. This will allow us to directly examine the nature of the conformational change, and the mechanisms that control the conformational change. It will also allow us to test the fundamentally novel hypothesis that differences in "structural flexibility" of the permeation pathway underlie, in part, differences in functional regulation of closely related ion channels. The ability to incorporate this technology into our research will provide a new and enhanced approach for our study of ion channel mechanisms, and will allow us to collect preliminary data necessary for subsequent funding.