Thesis (Ph. D.)--University of Chicago, Dept. of Neurobiology, Pharmacology and Physiology, August 2003. / Includes bibliographical references. Also available on the Internet.

Thesis (M.S.)--Ohio State University. / Includes bibliographical references (leaves 60-62). Available online via OhioLINK's ETD Center

RCK (regulating the conductance of K+) domains are ubiquitous among a wide variety of cation translocation systems or channels. Whether it is inward or outward transport, RCK domains share high sequence similarity from protein to protein, suggesting that they perform important functions in these systems. Although specific functions are not yet fully understood, RCK domains bind nucleotides, a characteristic that has been suggested to be important for the open/closed transition. In some cases RCK domains can bind additional ligands, e.g. KefC binding to glutathione. This project provides an in-depth study of two equally important bacterial potassium uptake systems, Trk (from E. coli) and CglK (from C. glutamicum). In these systems, RCK domains form octamers that either are anchored (CglK) or are separate and bind to transmembrane partners (Trk). The overall objective of this study was to examine the ligand control of the Trk potassium uptake system, including ligand identification and binding effects on its conformation, and therefore activity control. A crystal structure of the Trk potassium uptake system from Vibrio parahaemolyticus, was published recently [1] and provided evidence of ADP/ATP switch as a control mechanism. In accordance with the aforementioned study, the work presented in this thesis provides strong evidence that both ADP and ATP can bind to E. coli TrkA. Furthermore, it was shown that NADH bind strongly to one of the two RCK domains present in TrkA protein. The data presented here suggest a more complex control mechanism of the E. coli Trk system. CglK is a major potassium uptake system of C. glutamicum, but little is known about its control mechanism. The mutagenesis approach was undertaken to learn more about the system and its underlying processes/mechanisms. The work shown in this thesis indicates a similarity of CglK to other potassium channels, such as MthK and GsuK. The functional mechanisms proposed for those two systems were proven to be similar to that of CglK, although the specific CglK activation ligands are still to be found.

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Two kinetically and pharmacologically distinct outward K+ conductances were found to be simultaneously activated at the plasma membrane of TBY-2 suspension culture protoplasts using the whole-cell patch-clamp technique. We used a modified Hodgkin-Huxley model to quantify the activation kinetics of the outward current. Time constants for the two conductances derived from the model differed in magnitude by 5--10 fold over the voltage range from -10 mV to +50 mV, allowing their classification as either fast or slow. Deactivation kinetics were better fit by two exponential terms rather than one, yielding fast and slow deactivation time constants. The voltage dependence of time constants derived from these two independent two-channel models followed a bell-shaped distribution with mid-point potentials for both components at -20 mV with a standard 10-fold K+ gradient (10 K+o/100K+i). / Both components were highly K+-selective, however the tail current amplitudes of the slowly activating component at hyperpolarized potentials exhibited non-linear rectification whereas the tail current amplitudes of the fast activating component were linear. The ratio of inward tail current/activated outward current (envelope of tails test) was not constant during the depolarizing step; during the first 50--100 milliseconds the ratio was 6 times higher than at quasi-steady-state (i.e. after 0.3 second). / A pharmacological dissection of outward currents revealed that external Ba2+ in the range from 10 muM to 1 mM selectively inhibited a fast, sigmoidally activating, slowly inactivating current as revealed by examining difference currents. The more slowly activating component was inhibited by only 20% with 5 mM Ba2+. Conversely, nitrendipine or bepridil (5--100 muM) selectively inhibited the slower component of outward current. External TEA inhibited both the fast and slow components equally; tail current amplitudes of both components were inhibited by 40% with 2 mM TEA and the activation time courses in the presence of TEA conserved the same kinetic parameters as control currents.

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Voltage clamp fluorometry (VCF) was first developed in the mid 1990s by Isacoff and his colleagues. In this approach fluorophores are attached to substituted cysteine residues that are engineered by site-directed mutagenesis. Changes in the dielectric environment of the fluorophore report local transitions that are associated with electrically-related and electrically-silent transitions. VCF provides a powerful technique to observe real time reports of ion channel gating conformations. It has proven to be a useful technique because it adds insight that is not available using other techniques. X-ray crystallography studies give a predominantly static picture of the channel, while patch clamping of channels gives information only about residues that effect ionic current flow. Similarly, gating current provides insight only about residues that are charged and move across the membrane electric field. In this thesis we examined the structural rearrangements of the Shaker channel and the effect of 4-AP on channel gating. We also examined for the first time the structural rearrangements of the Kv1.5 gating and the how the channel responds to depolarization pulses. This work is instrumental in the examination of the potassium channel gating.