Karl L. Magleby - US grants

The overall purpose of this research proposal is to determine the properties and underlying kinetic mechanisms of two different ionic channels in cell membranes: the calcium-activated potassium channel and a voltage dependent chloride channel of large conductance. Currents that flow through single ionic channels in the sarcolemma of cultured rat skeletal muscle cells will be recorded under voltage clamp using the patch clamp technique. With this electrophysiological technique it is possible to study the kinetics of single channels by observing (through step changes in the current) when single channels open and close. The effects of Na, K, and Mg ions, step changes in [Ca] ion, and possible phosphorylation on the activity of the Ca-activated K channel will be characterized. The mechanism underlying the observation that openings of the Ca-activated K channel tend to occur in bursts will also be examined. The effect of membrane potential on activation, inactivation, and removal of inactivation for the voltage dependent C1 channel of large conduictance will also be characteized, as well as the permeability of this channelto various ions. The above data will be used to obtain information about the number of open and closed states, the mean lifetimes of the open and closed states, and the rate constants for transitions between the various states for each channel. These findings will then be used to develop a kinetic scheme for each channel. The Ca-activated K channel modulates repetitive firing in neurons and affects membrane potential and excitability in muscle. The C1 channel of large conductance would also affect membrane excitability. To understand the properties and function of nerve and muscle membranes, it will be necessary to understand the properties and mechanisms of these two channels. Understanding these channels may also help in understanding and treating nerve and muscle disease, as therapeutic agents and toxins often exert their specific effects on ionic channels in cell membranes.

DESCRIPTION (provided by applicant): The long term objectives of this research are to understand the mechanisms by which ion channels gate their pores. Research in this proposal focuses on the large conductance Ca2+ and voltage-activated (BK) channel, which plays a key role in many physiological functions, including control of muscle contraction, regulation of neuronal excitability, and control of transmitter release. BK channels are tetramers, with one voltage sensor, two high affinity Ca2+ sensors, and one low affinity Ca2+ sensor on each of the four subunits. Although much progress has been made towards understanding the contributions of these different sensors in activating the channel, a comprehensive kinetic mechanism to describe gating at the single-channel level is not yet available. To develop this mechanism, channels will be expressed in HEK 293 cells and Xenopus oocytes, and currents will be recorded from single BK channels in excised patches of membrane using the patch clamp technique. The single-channel data will then be analyzed by simultaneously fitting two-dimensional dwell-time distributions of adjacent open and closed interval durations obtained over wide ranges of Ca2+ and voltage to determine the underlying gating mechanism. Aim 1 will isolate and characterize the contribution of each of the three types of Ca2+ sensors to the gating. This will be done in the presence of the voltage sensors to characterize possible interactions among the various sensors. The hypothesis to be tested is that the gating of BK channels modified to have one type of Ca2+ sensor and one voltage sensor per subunit will be described by two-tiered 50 state allosteric gating mechanisms. Aim 2 will use the information obtained in Aim 1 together with additional experimental information to develop a comprehensive kinetic gating mechanism for wild type BK channels with their full complement of one voltage sensor and three different Ca2+ sensors per subunit. The hypothesis to be tested is that the Ca2+ and voltage dependent gating of BK channels is consistent with large two-tiered, recursive, 1250 state allosteric gating mechanisms. The kinetic gating mechanisms to be developed will specify the number of states the channel enters during gating, the transition pathways among the states, the rate constants for the transitions, the voltage and Ca2+ dependence of the rate constants, the allosteric changes in the opening and closings rates for each activated sensor, and the interactions among the various sensors. The ability of the model to describe gating will be tested for single-channel currents and also for published macroscopic ionic and gating currents. Defective BK channels are associated with hypertension, bladder disorder, epilepsy, paroxysmal movement disorder, autism, and mental retardation. The information to be obtained about gating mechanism should be useful towards identifying and understanding disease processes associated with BK channels and designing therapeutic interventions to restore disrupted physiological function. PUBLIC HEALTH RELEVANCE: Large conductance calcium and voltage activated potassium (BK) channels are involved in many key physiological processes including controlling skeletal and smooth muscle contraction, regulating the excitability of nerve cells, and modulating hormone and transmitter release. Defective or missing BK channels have been implicated in hypertension, bladder disorders, epilepsy, paroxysmal movement disorder, autism, and mental retardation. The proposed studies will provide insight into how BK channels function, which will be useful towards understanding disease processes associated with BK channels and in the development of therapeutic interventions to restore disrupted physiological function.

This is a proposal to acquire an atomic force microscope (AFM) on an inverted optical microscope and to develop two AFM-related non-imaging instruments: one for measuring single-molecule force spectroscopy and inter-molecular forces; the other, for measuring elasticities of soft samples under physiological conditions at the nano-scale. Over the past 10 years, atomic force microscopy (AFM) has become an increasingly important tool in biological research. It has gained popularity in biological applications because, unlike electron microscopy, it can image samples under physiological conditions, including live cells undergoing biological processes. The AFM acquires a topographical image of the sample surface by raster scanning an atomically sharp probe over the sample. In addition to its different imaging modes, the AFM is a versatile instrument that can be applied as a nano-indenter and as a molecular force apparatus to probe the mechanical properties of the sample. As a nano-indenter, the AFM has provided direct measurements of the local viscoelastic properties of samples on the nanometer scale. As a molecular force apparatus, the AFM has been used to measure the unbinding force of individual ligand-receptor complexes and the unfolding of individual proteins. Another attractive feature of the AFM is that it can be readily combined with optical microscopy techniques such as FRET, FRAP, TIRF and confocal microscopy. By integrating optical microscopy and AFM into a single experimental platform, the optical image can be directly correlated with the AFM data, providing a powerful tool for studying biological process in situ and in real time.

The acquisition and development of these three instruments is the first step toward establishing an ultramicroscopy center at the university. The two instruments to be developed can be constructed very economically, based on the designs of existing AFMs from the principal investigator's laboratory; this will permit the commercial AFM to be dedicated to imaging applications. The commercial AFM will be the first imaging AFM in the South Florida area and will provide a much needed resource for the local research community. These instruments will provide valuable research opportunities for undergraduates and students from underrepresented groups as well as researchers from different disciplines within the university.

? DESCRIPTION (provided by applicant): High conductance Ca2+ and voltage activated K+ channels (Slo1 or BK channels) are widely distributed and play numerous physiological roles. BK channels function as ? subunits alone, as in skeletal muscle, or in association with auxiliary ? subunits (?1- ?4) where they confer diverse functional properties in different tissues. Defectve or missing BK channels or their ? subunits have been associated with many disease processes including hypertension, asthma, autism, mental retardation, obesity, and epilepsy. Understanding the normal mechanism of activation of BK channels is crucial to understanding how function is altered in disease, and to provide molecular information that would be useful in developing possible therapies. The four ? subunits of Slo1 assemble to form a channel with an intra-membrane Core and a cytoplasmic Tail. The Core consists of four voltage sensing domains (VSD) and a pore gate domain (PGD). The cytoplasmic Tails form a large intracellular gating ring. Ca2+ binding to the gating ring activates the PGD through a poorly understood coupling mechanism from gating ring to Core. Also poorly understood are the sites and mechanisms of action of the various ? subunits on BK channels. Two recent advances will allow us to apply new approaches to resolve the mechanisms of coupling and ? subunit action. The first advance is obtaining the protein crystal structures of the gating ring in the closed and open conformations, which suggests that Ca2+ binding to the gating ring induces a push to the Core under the VSDs resulting from elevation of the four alpha-B helices of the gating ring, and a simultaneous pull on the S6 segments in the PGD of the Core arising from movement of lever arms in the gating ring. The second advance was our isolation and functional expression of the isolated Core itself, which provides a tool to assign observed functions to Core, gating ring, or both. Based on these advances and functional data we hypothesize that a novel Push-Pull mechanism couples Ca2+-dependent activation from the gating ring to the Core. In Aim 1 we critically test the Push-Pull hypothesis for Ca2+-dependent coupling using mutations with expected outcomes based on the Push-Pull hypothesis. In Aim 2 we use these advances to localize the sites of action of ?1- ?4 subunits on modifying gating of BK channels to the Core, gating ring, or both. In Aim 3 we seek to obtain the protein crystal structures of the mutated gating rings that alter Ca2+-dependent coupling and also the structures of the sites of contact between peptides of ? subunits and gating ring to provide structural insight into mechanism. The completion of these aims should provide new insight into the mechanism of Ca2+-dependent coupling between gating ring and Core, and also into the mechanisms for ? subunit modulation of BK channels. The Push-Pull model, if found to be consistent with the critical tests to be applied, will necessitate a paradigm shift in the proposed mechanism of Ca2+-dependent coupling, from a single active coupling structure to dual simultaneously active Push-Pull coupling structures.