Daniel H. Cox - US grants

Large conductance Ca2+-activated K+ channels (BKCa, channels), provide feedback control for a number Ca2+-dependent physiological processes. In some tissues these channels may be composed simply of four a subunits surrounding a central pore. In smooth muscle, however, the BKCa channel's Ca2+ sensitivity is greatly enhanced and its kinetic behavior is altered by an auxiliary subunit known as beta1. These effects are essential for the channel to function properly in smooth muscle. Despite the BKCa channel's physiological importance and the powerful modulatory effects of beta1, until recently very little was known about the mechanism by which beta1 alters channel behavior. By performing experiments proposed in the original submission of this application, however, we have gone some distance addressing this issue. Specifically, we have found that despite beta1's large effects on Ca2+ sensitivity, beta1 appears to alter Ca2+ binding very little. Instead, other aspects of gating are altered by beta1, aspects that indirectly influence Ca2+ sensitivity through altering the voltage-dependent mechanism of channel gating. Having come to this understanding, under Specific Aim 1 of the present application we propose to examine quantitatively how beta1 alters BKCa voltage-dependent gating so as to gain further insight into the biophysical mechanism behind beta1's ability to enhance Ca2+ sensitivity. In Specific Aim 2 we then turn our attention to beta1's molecular mechanism, addressing the question: what regions of beta1 make functionally important interactions with the BKCa a subunit? We view these experiments as an important step toward elucidating the molecular interactions involved in B-mediated BKCa channel regulation. We also propose in Specific Aim 3 experiments to determine whether different types of BKCa beta subunits (four beta subunits have now been identified beta1, beta2, beta3, beta4) can associate with a single channel and, as well, to examine the properties of BKCa channels coupled to differing numbers of beta1 subunits. This will allow us to test the hypothesis that variation in subunit stoichiometry contributes to the functional heterogeneity of BKCa channels in native tissues. Information for the proposed experiments will advance our understanding of the relationship between BKCa channel structure and function and as well the regulation of these channels in vivo. It may also form the basis for the development of therapeutic agents that modulate BKCa channel gating.

DESCRIPTION (provided by applicant): The large conductance Ca2+-activated K+ channel, or BKCa channel, is found in a wide variety of cell types where in general it provides feedback control over processes like neurotransmission and smooth muscle contraction that involve membrane-potential depolarization and a rise in intracellular Ca2+. Despite, however, its physiological importance, still a great deal remains to be understood about how this channel senses and responds to its primary stimuli Ca2+ and membrane voltage. In this application we propose experiments that will help remedy this situation. Three Aims are proposed. In Aim 1 we will test the prevailing hypothesis that the Ca2+-sensing mechanism of the BKCa channel is structurally similar to that of the bacterial Ca2+-activated K+ channel MthK, for which there is a crystal structure. In Aim 2, we will use electrophysiological recordings to comprehensively examine the energetics of BKCa Ca2+ sensing, and in Aim 3 we will take advantage of a recent technical advance to examine the movement of the BKCa- channel's voltage sensors exclusively when the channel is open. Results from these experiments will greatly advance our understanding of the regulation of this physiological important protein, and in so far as understanding precedes control, they may also aide in the development of pharmacological agents to be used to modulate the important physiological processes these channels regulate. PUBLIC HEALTH RELEVANCE: We propose here a series of studies that will greatly advance the understanding of the regulation of a particular ion channel protein, the large-conductance calcium-activated potassium channel, or BK channel, which plays a vital role in the regulation of neurotransmitter release in the brain and smooth-muscle contraction in the vasculature, the reproductive and digestive systems and the lungs. Because of its physiological importance, pharmacological agents that open this channel are currently being sought for the treatment of such diverse conditions as high blood pressure, stroke, asthma, impotence and incontinence. As the work proposed here is designed to uncover the natural mechanisms by which this channel is stimulated to open, it may very well point the way to the development of such beneficial pharmaceuticals.