Pharmacology and pore-forming domains of the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel member of the ATP-binding cassette (ABC) superfamily of membrane proteins. CFTR has two homologous halves, each consisting of six transmembrane spanning domains (TM) followed by a nucleotide binding fold, connected by a regulatory (R) domain. This thesis addresses the question of which domains are responsible for Cl^- selectivity, i.e., which domains line the channel pore.

To address this question, novel blockers of CFTR were characterized. CFTR was heterologously expressed in Xenopus oocytes to study the mechanism of block by two closely related arylaminobenzoates, diphenylamine-2-carboxylic acid (DPC) and flufenamic acid (FFA). Block by both is voltage-dependent, with a binding site ≈ 40% through the electric field of the membrane. DPC and FFA can both reach their binding site from either side of the membrane to produce a flickering block of CFTR single channels. In addition, DPC block is influenced by Cl^- concentration, and DPC blocks with a bimolecular forward binding rate and a unimolecular dissociation rate. Therefore, DPC and FFA are open-channel blockers of CFTR, and a residue of CFTR whose mutation affects their binding must line the pore.

Screening of site-directed mutants for altered DPC binding affinity reveals that TM-6 and TM-12 line the pore. Mutation of residue 5341 in TM-6 abolishes most DPC block, greatly reduces single-channel conductance, and alters the direction of current rectification. Additional residues are found in TM-6 (K335) and TM-12 (T1134) whose mutations weaken or strengthen DPC block; other mutations move the DPC binding site from TM-6 to TM-12. The strengthened block and lower conductance due to mutation T1134F is quantitated at the single-channel level. The geometry of DPC and of the residues mutated suggest α-helical structures for TM-6 and TM-12. Evidence is presented that the effects of the mutations are due to direct side-chain interaction, and not to allosteric effects propagated through the protein. Mutations are also made in TM-11, including mutation S1118F, which gives voltage-dependent current relaxations. The results may guide future studies on permeation through ABC transporters and through other Cl^- channels.

Structural and Biophysical Characterization of Variants of the Mechanosensitive Channel of Large Conductance (MSCL)

The ability to sense mechanical force is vital to all organisms to interact with and respond to stimuli in their environment. Mechanosensation is critical to many physiological functions such as the senses of hearing and touch in animals, gravitropism in plants and osmoregulation in bacteria. Of these processes, the best understood at the molecular level involve bacterial mechanosensitive channels. Under hypo-osmotic stress, bacteria are able to alleviate turgor pressure through mechanosensitive channels that gate directly in response to tension in the membrane lipid bilayer. A key participant in this response is the mechanosensitive channel of large conductance (MscL), a non-selective channel with a high conductance of ~3 nS that gates at tensions close to the membrane lytic tension.

It has been appreciated since the original discovery by C. Kung that the small subunit size (~130 to 160 residues) and the high conductance necessitate that MscL forms a homo-oligomeric channel. Over the past 20 years of study, the proposed oligomeric state of MscL has ranged from monomer to hexamer. Oligomeric state has been shown to vary between MscL homologues and is influenced by lipid/detergent environment. In this thesis, we report the creation of a chimera library to systematically survey the correlation between MscL sequence and oligomeric state to identify the sequence determinants of oligomeric state. Our results demonstrate that although there is no combination of sequences uniquely associated with a given oligomeric state (or mixture of oligomeric states), there are significant correlations. In the quest to characterize the oligomeric state of MscL, an exciting discovery was made about the dynamic nature of the MscL complex. We found that in detergent solution, under mild heating conditions (37 °C – 60 °C), subunits of MscL can exchange between complexes, and the dynamics of this process are sensitive to the protein sequence.

Extensive efforts were made to produce high diffraction quality crystals of MscL for the determination of a high resolution X-ray crystal structure of a full length channel. The surface entropy reduction strategy was applied to the design of S. aureus MscL variants and while the strategy appears to have improved the crystallizability of S. aureus MscL, unfortunately the diffraction qualities of these crystals were not significantly improved. MscL chimeras were also screened for crystallization in various solubilization detergents, but also failed to yield high quality crystals.

MscL is a fascinating protein and continues to serve as a model system for the study of the structural and functional properties of mechanosensitive channels. Further characterization of the MscL chimera library will offer more insight into the characteristics of the channel. Of particular interest are the functional characterization of the chimeras and the exploration of the physiological relevance of intercomplex subunit exchange.

The equivalent conductance of solutions of picric acid

Picric acid possesses the property, which is rare among strong electrolytes, of having a convenient distribution ratio between water and certain organic solvents such as benzene, chloroform, etc. Because of this property, picric acid offers peculiar advantages for studying the well known deviations of strong electrolytes from the law of mass action, for; by means of distribution experiments, the activities of picric acid in various aqueous solutions may be compared.

In order to interpret the results of such distribution experiments, it is necessary to know the degree of ionization of picric acid in aqueous solutions.

At least three series of determinations of the equivalent conductance of picric acid have been published, but the results are not concordant; and therefore, the degree of ionization cannot be calculated with any degree of certainty.

The object of the present investigation was to redetermine the conductance of picric acid solutions in order to obtain satisfactory data from which the degrees of ionization of its solutions might be calculated.

Conductance calculation of a long tube with equilateral triangle cross section

Using the stratified gas flow model for calculating the conductance of long tubes with constant cross section, an analytical expression for calculating the conductance of along tube with equilateral triangle cross section has been derived. The formula given is applicable to the full pressure range. A minimum in the conductance in the intermediate flow state is shown. 2002 American vacuum Society.

ac gate-induced Fano resonance and peak splitting of conductance in a quantum dot

We have investigated the conductance of a quantum dot system suffering an anti-symmetric ac gate voltage which induces the transition between dot levels in the linear regime at zero temperature in the rotating wave approximation. Interesting Fano resonances appear on one side of the displaced resonant tunnelling peaks for the nonresonant case or the peak splitting for the resonant case. The line shape of conductance (vs Fermi energy) near each level of the quantum dot can be decomposed into two profiles: a Breit-Wigner peak and a Fano profile, or a Breit-Wigner peak and a dip in both cases.

Reflected in-plane conductance near a barrier with Dresselhaus spin-orbit coupling

We have calculated the in-plane conductance of a barrier with the Dresselhaus spin-orbit interaction, which is sandwiched between two spin-polarized materials aligned arbitrarily. Besides a transmitted in-plane current which arises on the drain side as pointed out in Phys. Rev. Lett. 93, 056601 (2004), a reflected in-plane current always appears simultaneously on the source side near the interface of the barrier. The spin polarization of the source affects the transmitted current more than the reflected one, and conversely the spin polarization of the drain affects the reflected current more. The relationship between transmitted current and the reflected one has been studied.

Interdot interaction induced zero-bias maximum of the differential conductance in parallel double quantum dots

We have studied the equilibrium and nonequilibrium electronic transports through a double quantum dot coupled to leads in a symmetrical parallel configuration in the presence of both the inter- and the intradot Coulomb interactions. The influences of the interdot interaction and the difference between dot levels on the local density of states (LDOS) and the differential conductance are paid special attention. We find an interesting zero-bias maximum of the differential conductance induced by the interdot interaction, which can be interpreted in terms of the LDOS of the two dots. Due to the presence of the interdot interaction, the LDOS peaks around the dot levels epsilon(i) are split, and as a result, the most active energy level which supports the transport is shifted near to the Fermi level of the leads in the equilibrium situation. (c) 2006 American Institute of Physics.

Mechanisms for extra conductance plateaus in quantum wires

Spin-density-functional theory is employed to calculate the conductance G through a quasi-one-dimensional quantum wire. In addition to the usual subband quantization plateaus at G=n(2e(2)/h), we find additional structures at (n+1/2)(2e(2)/h). The extra structures appear whether or not the electrons in the wire spin polarize. However, only the spin-polarized case reproduces the experimental temperature and magnetic field dependences.

Oscillatory magneto-conductance in quantum waveguides with lateral multi-barrier structures

The magneto-transport properties of a narrow quantum waveguide with lateral multibarrier modulation are investigated theoretically. It is found that the magnetoconductance as a function of Fermi energy or magnetic field exhibits square-wave-like oscillations. In the presence of magnetic field, the edge states are formed near each barrier and the boundaries. Therefore, the number of edge states increases with the number of lateral barriers, leading to the increase of the propagating modes. On the other hand, owing to the tunneling effect a pair of edge states around the barrier region with opposite moving directions may be coupled and formed a circulating localized state, leading to the quenching of the related propagating states. The resulting dispersion relation exhibits oscillation structures superimposed on the bulk Landau levels. These novel conductance characteristics may provide potential applications to the fabrication of new quantum devices.