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Ras proteins activate calcium channels in neuronal cells
J Hescheler1, F J Klinz, G Schultz
1Pharmakologisches Institut der Freien Universität Berlin, Germany.
Abstract:
Ras (p21) proteins are involved in the control of cell growth and differentiation, but the mechanism by which they exert these effects is not yet known. Here we present evidence that c-Ha-ras (p21(Gly-12)) and its oncogenic mutant T24-ras (p21(Val-12)) selectively induce omega-conotoxin and dihydropyridine-sensitive Ca2+ currents within a few hours after introduction into the cytoplasm of neuroblastoma x glioma hybrid cells. Whereas control cells exhibited a mean Ca2+ current of 250 pA, it amounted to 730 pA in cells pretreated with ras protein. In cells loaded with p21(Gly-12), the effect occurred after 2 hours and was terminated after 8 hours. In contrast, introduction of p21(Val-12) resulted in a prolonged delay (6 hours) of the effect which lasted for more than 24 hours. When ras proteins were preactivated with the non-hydrolysable GTP analog GppNHp, the time courses of both p21(Gly-12) and p21(Val-12) effects were fast and sustained, suggesting that in intact cells (i) the GDP/GTP exchange is faster for p21(Gly-12) compared to p21(Val-12) and (ii) inactivation of p21(Gly-12) is mediated by GAP-induced GTPase activity. T-type Ca2+ currents and K+ currents were unaffected by ras proteins.
Insights
Ras proteins regulate cell growth. This study shows Ras proteins selectively induce specific calcium (Ca2+) currents in hybrid cells, with differing time courses for normal and oncogenic forms.
Area of Science:
- Molecular Biology
- Cell Biology
- Neuroscience
Background:
- Ras proteins (p21) are key regulators of cell growth and differentiation.
- The precise mechanisms of Ras protein action remain largely unknown.
Purpose of the Study:
- To investigate the effect of Ras proteins on ion channel activity in neuroblastoma x glioma hybrid cells.
- To elucidate the role of Ras protein isoforms and their activation states in modulating calcium currents.
Main Methods:
- Introduction of c-Ha-ras (p21(Gly-12)) and T24-ras (p21(Val-12)) into hybrid cells.
- Measurement of omega-conotoxin and dihydropyridine-sensitive Ca2+ currents using patch-clamp electrophysiology.
- Preactivation of Ras proteins with GppNHp to assess the role of GTP/GDP exchange.
Main Results:
- Ras proteins selectively induced Ca2+ currents, increasing them from 250 pA to 730 pA.
- p21(Gly-12) induced currents rapidly (2 hours) with short duration (8 hours).
- p21(Val-12) induced currents with a delayed onset (6 hours) and prolonged duration (>24 hours).
- Preactivated Ras proteins showed fast and sustained current induction, indicating differential GDP/GTP exchange and inactivation rates.
Conclusions:
- Ras proteins modulate specific calcium channel activity.
- The kinetics of Ras protein signaling differ between normal and oncogenic forms, influencing downstream effects.
- Ras protein inactivation is likely mediated by GTPase-activating protein (GAP) activity.