Stable Expression of Gastric Proton Pump Activity at the Cell Surface

Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.

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Beta-adrenergic modulation of Na-K pump activity in young and adult canine cardiac Purkinje fibers

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1996.271.2.h706 ◽

1996 ◽

Vol 271 (2) ◽

pp. H706-H712

Author(s):

F. Charpentier ◽

M. J. Legato ◽

S. F. Steinberg ◽

I. S. Cohen ◽

M. R. Rosen

Keyword(s):

Cell Surface ◽

Volume Ratio ◽

Beta Adrenergic ◽

Purkinje Fibers ◽

Age Related ◽

Cardiac Purkinje Fibers ◽

Adrenergic Modulation ◽

Pump Activity ◽

The Difference ◽

Beta Adrenergic Agonist

We used standard microelectrode techniques to study the developmental changes and beta-adrenergic modulation of membrane potential and of Na-K pump activity in adult (> 1 yr of age) and neonatal (2-10 days) canine Purkinje fibers. Isoproterenol (10(-7) M) increased the rate of development and magnitude of pacing-induced hyperpolarization of adult fibers driven at a 1-s cycle length. This effect of isoproterenol was attenuated by treating dogs with pertussis toxin (PTX), (30 micrograms/kg). Other adult and neonatal fibers were superfused with a Tyrode solution containing Ba2+ 0.2 mM, Cs+ 2 mM, and 10(-6) M verapamil, thus leading to depolarization and cessation of spontaneous activity. The Na-K pump was studied by alternating solutions containing [K] at 0 mM (inhibiting the pump) and 4 mM (reactivating the pump). Although the kinetics of the Na-K pump appeared faster in neonatal fibers than in adult fibers, measurement of cell surface-to-volume ratio compensated for the difference. We therefore conclude that 1) the apparent age-related changes in Na-K pump activity in canine Purkinje fibers in fact reflect cell surface-to-volume ratio and, 2) the beta-adrenergic agonist-induced hyperpolarization in adults requires the presence of a PTX-sensitive G protein for its occurrence.

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Sites of resistance changes with inhibition of acid secretion in frog stomach

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1986.250.5.g639 ◽

1986 ◽

Vol 250 (5) ◽

pp. G639-G647

Author(s):

W. S. Rehm ◽

G. Carrasquer ◽

M. Schwartz

Keyword(s):

Acid Secretion ◽

Proton Pump ◽

Tubular Cell ◽

Secretory Rate ◽

Tubular Cells ◽

Resistance Increase ◽

Frog Stomach ◽

Scn Resistance ◽

Gastric Proton Pump ◽

Resistance Decrease

The primary purpose of this study was to determine the sites of resistance changes from thiocyanate inhibition of secretion to better evaluate models of the gastric proton pump. The potential difference, resistance, and H+ secretory rate were measured with various tonicities of the secretory fluid. Thiocyanate (SCN) inhibition generally produces a resistance increase, but with nutrient Ba, the resistance is high and SCN decreases it. The latter effect is also seen with cimetidine. In the secreting fundus a hypotonic secretory solution has a small effect on resistance, but in the inhibited fundus the effect is huge, due to increase of resistance of the lumen-tubular cell pathway. With a hypotonic secretory solution, for inhibited fundus (cimetidine or omeprazole) and antrum, SCN does not decrease the resistance of the surface cells and/or the transintercellular pathways with or without Ba. The SCN resistance decrease with Ba is via the tubular cell pathway. With Ba the resistance of the nutrient membrane of the tubular cells decreases after SCN or cimetidine inhibition. The findings further support the concept that under standard conditions the resistance via the lumina and tubular cells is low and that via the surface cells and transintercellular pathways is high.

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