Kinetic Properties of DM-Nitrophen Binding to Calcium and Magnesium

Abstract

Caged-Ca(2+) compounds such as nitrophenyl-EGTA (NP-EGTA) and DM-nitrophen (DMn) are extremely useful in biological research, but their use in live cells is hampered by cytoplasmic [Mg(2+)]. We determined the properties of Ca(2+) release from NP-EGTA and DMn by using Oregon green BAPTA-5N to measure changes in [Ca(2+)] after ultraviolet flash photolysis in vitro, with or without Mg(2+) present. A large fraction (65%) of NP-EGTA, which has a negligible Mg(2+) affinity, uncages with a time constant of 10.3 ms, resulting in relatively slow increases in [Ca(2+)]. Uncaging of DMn is considerably faster, but DMn has a significant affinity for Mg(2+) to complicate the uncaging process. With experimentally determined values for the Ca(2+) and Mg(2+) binding/unbinding rates of DMn and NP-EGTA, we built a mathematical model to assess the utility of NP-EGTA and DMn in rapid Ca(2+)-uncaging experiments in the presence of Mg(2+). We discuss the advantages and disadvantages of using each compound under different conditions. To determine the kinetics of Ca(2+) binding to biologically relevant Ca(2+) buffers, such as Ca(2+)-binding proteins, the use of DMn is preferable even in the presence of Mg(2+).

Readily releasable pool of synaptic vesicles measured at single synaptic contacts

“…In the axon, the fluorescence change, ΔF/F 0 , due to Ca 2+ release by laser pulse photolysis (Fig. 2) showed a sharp rise in <1 ms, consistent with rapid Ca 2+ release by photolysis of DM-nitrophen-Ca 2+ , which has been reported to have a 15-μs time constant (26). The mean decay time constant of Ca 2+ -induced fluorescence was 120.7 ± 63.5 ms (mean ± SD; n = 49 regions of interest from 18 different cells; single laser pulses 0.1-1 ms in duration, energies 0.2-5 μJ at the cell).…”

The continuing disappearance of “pure” Ca2+ buffers

“…The most detailed in vitro study on the binding parameters of CR was carried out by applying the same methodology, i.e., release of caged Ca 2+ from DM-nitrophen [174], as used before for the determination of the binding properties of CB-D28k [125]. The prior determination of the kinetic properties of DM-nitrophen, i.e., its binding to Ca 2+ and Mg 2+ [175] was a requirement for the accurate modeling of CR. Faas and co-workers [174] developed a new mathematical model to determine the dynamic properties of cooperative binding to gain a better understanding of the physiological implications of cooperativity.…”

The Contribution of RCK Domains to Human BK Channel Allosteric Activation

“…We combined the voltage-clamp fluorometry technique (40 -42) with the UV photolysis of a Ca 2ϩ cage compound, DM-Nitrophen (43)(44)(45)(46), to simultaneously trigger voltage-and Ca 2ϩ -dependent activations of BK channels. We found that rapid intracellular Ca 2ϩ release produced structural rearrangements that not only increased channel P o but also perturbed the conformation of the S4 helix of the BK VSD.…”