PubMed · 10277673
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C F Kerchner. 1986. Power line problems.. https://pubmed.ncbi.nlm.nih.gov/10277673/
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The activation kinetics of outward currents in protoplasts from barley root xylem parenchyma was investigated using the patch-clamp technique. The K(+) outward rectifying conductance (KORC), providing the main pathway for K(+) transport to the xylem, could be described in terms of a Hodgkin-Huxley model with four independent gates. Gating of KORC depended on voltage and the external K(+) concentration. An increase in the external K(+) concentration resulted in a shift in the voltage dependence of gating. This could be explained by a K(+) dependence of the rate constant beta for channel closure, indicating binding of K(+) to a regulatory site exposed to the bath. Occasionally, KORC was observed to inactivate; this inactivation occurred and vanished spontaneously. In some of the whole cell and excised patch recordings, a stepwise increase in outward current was observed upon a depolarizing voltage pulse, indicating that several populations of 'sleepy' channels existed in the plasma membrane that activated with a certain lag time. It is discussed whether this observation can be explained by a putative subunit, which retards channel activation, or by a scheme of cooperative gating. A quantitative description of outward rectifying K(+) channels in xylem parenchyma cells is a major step forward towards a mathematical model of salt transport into the xylem.
The porin from Paracoccus denitrificans, a slightly anion specific outer membrane pore protein, was expressed in Escherichia coli, isolated from inclusion bodies, and refolded in the presence of urea and detergents. The purified recombinant protein was reconstituted into black lipid bilayer membranes and showed no difference in its functional properties in comparison to the native porin isolated from P.denitrificans membranes. To investigate the molecular basis of its ion selectivity and voltage-gating, a series of site-directed mutants was constructed, comprising acidic residues located on the third extracellular loop (L3), which forms the constriction zone of the channel, and basic residues along the opposing barrel wall. Measurements using zero-current membrane potentials indicated that the selectivity changed drastically from a slight anion to a distinct cation selectivity with the exchange of residues R29 and R31 by glutamate, whereas replacements on the L3 loop went largely unaffected. However, when assaying the voltage-dependent closure of channels, only mutations located on the L3 loop showed an effect, in contrast to the voltage-independent recombinant and native Paracoccus porin.
A new way of regulation of electroosmotic flow (EOF) in capillary zone electrophoresis (CZE) by external electric field has been developed. A set of three high-voltage power supplies is used to form a radial electric field across the capillary wall. One power supply is applied in the usual way as a driving force of CZE and EOF to the ends of the inner capillary compartment dipped into the electrode vessels and filled with background electrolyte. Two power supplies are connected to the ends of the outer low-conductivity coating of the capillary which is formed by the dispersion of copolymer of aniline and p-phenylenediamine in polystyrene matrix. The difference between electric potentials on the outer capillary surface and inside the capillary determines the voltage of radial electric field across the capillary wall and affects the electrokinetic potential at the solid-liquid interface inside the capillary. The effect of magnitude and polarity of external radial electric field on the flow rate of EOF, on the migration times of charged analytes and on the separation efficiency and resolution of CZE separations of synthetic oligopeptides, diglycine, triglycine and octapeptide fragments of human insulin was evaluated. Through the EOF control by external electric field the dynamic effective length of the capillary was obtained and the speed of analysis and resolution of CZE separations of peptide analytes could be optimized.