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First published online October 19, 2007
Journal of Experimental Biology 210, 3771-3779 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.008037

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G protein activation by uncaging of GTP--S in the leech giant glial cell

Ingo C. Hirth, Frank C. Britz and Joachim W. Deitmer^*

Abteilung für Allgemeine Zoologie, FB Biologie, TU Kaiserslautern, PO Box 3049, D-67653 Kaiserslautern, Germany

View larger version (39K): [in this window] [in a new window]   Fig. 1. Changes in membrane potential (Em) and membrane resistance following uncaging of GTP--S. (A) Membrane potential recording of a giant glial cell injected with caged GTP--S, following moderate UV pulses (UV), the first of which hyperpolarized the cell membrane. (B) Changes in membrane potential following UV pulses; after a first (filled circles) and a second (open circles) UV pulse onto GTP--S-injected cells and after UV pulses of non-injected cells (open triangles). (C) Membrane input resistance (Rinput) measured by injection of square constant current pulses (upper trace), leading to brief deflection of the membrane potential (lower trace) before and after uncaging of GTP--S by a UV pulse. The inset shows superimposed potential traces at enhanced time scale before and after the UV pulse (indicated by shaded areas), showing a loss of input resistance after uncaging of GTP--S (D). (E,F) UV pulses onto non-injected cells had no effect on membrane potential or membrane resistance. **P<0.01.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Uncaging of GTP--S increases the relative K+ conductance of the membrane. (A) Effects of Leydig neuron stimulation (LS) before and after a UV pulse (UV) to uncage GTP--S on the membrane potential. After the membrane hyperpolarization following uncaging of GTP--S, LS had no additional effect on the membrane potential, while raising the extracellular K+ concentration from 4 to 10 mmol l–1 evoked a large depolarization. The inset shows the effect of LS and UV pulses on a non-injected cell. (B) Effects of 3 µmol l–1 myomodulin (MMHir) and 10 mmol l–1 K+ before and after a UV pulse uncaging GTP--S on the membrane potential. (C–E) The membrane depolarization induced by 10 mmol l–1 K+ is larger (C,D), and faster rising (C,E), after uncaging of GTP--S, indicating an increased relative K+ conductance. *P<0.05. N represents the number of experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Inhibition of G proteins after injection of GDP-ß-S. (A) Change in holding current (Im) after injection of the non-hydrolysable inhibitor of G proteins GDP-ß-S (filled circles) and of GDP (open circles) of a glial cell voltage-clamped to different holding potentials (set near to the zero current potential of each cell). (B–E) Currents during hyperpolarizing voltage (Vh) steps before and after injection of GDP-ß-S (B) or GDP (D), from which the membrane conductance (Gm) was calculated (C,E). ***P<0.001.

View larger version (26K): [in this window] [in a new window]   Fig. 4. The effect of uncaging GTP--S on the cytosolic Ca2+. (A) Glial cell injected with the fluorescent Ca2+ indicator Oregon Green BAPTA-1 and caged GTP--S showed a transient increase in cytosolic Ca2+ (F) and a sustained membrane hyperpolarization following a first UV pulse (UV), while a second UV pulse remained without effect. Membrane depolarization induced by raising the extracellular K+ concentration from 4 to 40 mmol l–1 induced a Ca2+ transient of similar amplitude. (B) Effect of UV pulse and 40 mmol l–1 K+ on a cell not injected with GTP--S. (C) Summary of cytosolic Ca2+ changes as shown in A and B. (D) Changes in cytosolic Ca2+ and membrane potential following uncaging of GTP--S and during membrane depolarization in 40 mmol l–1 K+ after depletion of the intracellular Ca2+ stores with cyclopiazonic acid (CPA, 20 µmol l–1). While high-K+ still evoked a Ca2+ rise, the uncaging of GTP--S did not change the cytosolic Ca2+ after store depletion (E) but still elicited the sustained membrane hyperpolarization. (F) Injection of the Ca2+ chelator BAPTA into the glial cell did not suppress the membrane hyperpolarization following the uncaging of GTP--S by a UV pulse. **P<0.01.

View larger version (16K): [in this window] [in a new window]   Fig. 5. The effect of uncaging GTP--S on cytosolic Na+. (A) Recording of intracellular Na+ and membrane potential before and after uncaging GTP--S by a UV pulse. (B–D) Steady-state cytosolic Na+ concentration before (control) and during uncaging GTP--S (B), before (control) and after a UV pulse on non-injected cells (C), and in the absence (control) and presence of cyclopiazonic acid (CPA, 20 µmol l–1), before (control) and after uncaging GTP--S in the presence of CPA (D). **P<0.01.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Effect of uncaging inositol-trisphosphate (IP3) on cytosolic Ca2+ and Na+. (A) Uncaging IP3 with a UV pulse elicited a cytosolic Ca2+ rise (F) but no membrane potential change. A second UV pulse changed neither cytosolic Ca2+ nor membrane potential, and membrane depolarization in 40 mmol l–1 K+ still evoked a normal transient Ca2+ rise. (B,C) Summary of effects of uncaging IP3 and of 40 mmol l–1 K+ on cytosolic Ca2+ (B) and on the membrane potential (C). (D,E) Uncaging of IP3 elicited a reversible intracellular Na+ rise but no membrane potential change. A second UV pulse had no effect on either Na+ or membrane potential. *P<0.05.