Showing papers by "Kenneth A. Stauderman published in 2004"
TL;DR: It is concluded that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.
Abstract: Using whole-cell recording in Drosophila S2 cells, we characterized a Ca2+-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca2+-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20–50 pA at −110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing ∼300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ >> Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of ∼50 nM, and was also blocked by 20 μM SKF 96365 and by 20 μM 2-APB. At concentrations between 5 and 14 μM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.
85 citations
Patent•
03 Mar 2004
TL;DR: In this paper, methods for identifying agents that modulate intracellular calcium are presented, as well as methods of modulating calcium within cells and methods of identifying proteins involved in modulating intra-cell calcium.
Abstract: Methods are provided for identifying agents that modulate intracellular calcium. Also provided are methods of modulating calcium within cells and methods of identifying proteins involved in modulating intracellular calcium.
50 citations
TL;DR: The characterization of two modes of gating of human CaV2.1 channels, the slow mode and the fast mode, which shows different rates of inactivation and different steady-state inactivation depending on the β subtype.
Abstract: The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels and their modulation by the auxiliary β1b, β2e, β3a, and β4a subunits were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing human CaV2.1 channels. These calcium channels showed a complex modal gating, which is described in this and the following paper (Fellin, T., S. Luvisetto, M. Spagnolo, and D. Pietrobon. 2004. J. Gen. Physiol. 124:463–474). Here, we report the characterization of two modes of gating of human CaV2.1 channels, the slow mode and the fast mode. A channel in the two gating modes differs in mean closed times and latency to first opening (both longer in the slow mode), in voltage dependence of the open probability (larger depolarizations are necessary to open the channel in the slow mode), in kinetics of inactivation (slower in the slow mode), and voltage dependence of steady-state inactivation (occurring at less negative voltages in the slow mode). CaV2.1 channels containing any of the four β subtypes can gate in either the slow or the fast mode, with only minor differences in the rate constants of the transitions between closed and open states within each mode. In both modes, CaV2.1 channels display different rates of inactivation and different steady-state inactivation depending on the β subtype. The type of β subunit also modulates the relative occurrence of the slow and the fast gating mode of CaV2.1 channels; β3a promotes the fast mode, whereas β4a promotes the slow mode. The prevailing mode of gating of CaV2.1 channels lacking a β subunit is a gating mode in which the channel shows shorter mean open times, longer mean closed times, longer first latency, a much larger fraction of nulls, and activates at more positive voltages than in either the fast or slow mode.
41 citations
Patent•
03 Mar 2004
TL;DR: In this article, methods for identifying agents that modulate intracellular calcium are presented, as well as methods of modulating calcium within cells and methods of identifying proteins involved in modulating intra-cell calcium.
Abstract: Methods are provided for identifying agents that modulate intracellular calcium. Also provided are methods of modulating calcium within cells and methods of identifying proteins involved in modulating intracellular calcium.
2 citations
Patent•
03 Mar 2004
TL;DR: In this paper, a method for identifying proteines impliquees dans the modulation of le calcium intracellulaire was proposed, based on the Ladite invention, which concerne des methodes d'identification d'agents who permettent de moduler le calcium intra-cellulaire.
Abstract: L'invention concerne des methodes d'identification d'agents qui permettent de moduler le calcium intracellulaire. Ladite invention a aussi trait a des methodes de modulation du calcium au sein de cellules et a des methodes d'identification des proteines impliquees dans la modulation du calcium intracellulaire.