Protoplasts were prepared from two yeast strains P. rhodozyma (ATCC 24202) and K. fragilis (ATCC 8455). Protoplasts prepared from P. rhodozyma were facilitated by prior growth of the cells in a media containing S-(2-aminoethyl)-L-cysteine. Protoplasts from these two yeast genera were fused either by the use of electrofusion or polyethylene glycol treatment. Stable carotenoid producing cell lines were selected by growth at 30°C on yeast nitrogen base plus galactose. Selected single fusants display taxonomic characteristics common to both genera with a cellular morphology and a carotenoid composition similar to that of P. rhodozyma. / Land and Food Systems, Faculty of / Graduate

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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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Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 125-138). Also available via the Internet.

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