Indo-1 can simultaneously detect Ba2+ entry and Ca2+ blockade at a plasma membrane calcium channel

Abstract

The fluorescent chelator Indo-1 can make simultaneous determinations of two intracellular ion concentrations, such as [Ca2+] and [Cd2+], or [Ca2+] and [Ba2+], in a normal cell suspension. The second ion can be detected even if its spectrum when bound to Indo-1 is same as for the calcium-bound or the ion-free Indo-1, as long as there is a change in height. This is because the mathematical analysis uses not only the spectral shape, but also takes into account increases in total signal intensity. For maximum accuracy, whole spectra were analyzed. When 3 mM [Ba2+] was added to a B cell line that had been stimulated with antiimmunoglobulin to open receptor operated calcium channels, there was a sudden drop in 400 nm Indo-1 fluorescence. Spectral analysis showed that this was due to a drop in intracellular [Ca2+], which was consistent with blockage of the receptor-operated calcium current by extracellular Ba2+. The conductance for Ba2+ was also observable as a slow rise in total fluorescence. There was also a slow increase in intracellular [Ca2+] as barium accumulated in the cell, which was tentatively attributed to blockage of the plasma membrane calcium pump by intracellular Ba2+.

Extracellular calcium sensing promotes human B-cell activation and function

“…51 The role of calcium channels in Ca 2ϩ o sensing in B cells was further excluded as channel inhibitors, such as SKF96365, econazole, and verapamil, did not inhibit Ca 2ϩ o -induced Ca 2ϩ i release or CD83 expression (data not shown). In addition, the presence of equimolar concentrations of BaCl 2 in lieu of CaCl 2 did not induce changes in the fluorescence of Indo-1 AM, further excluding a role for calcium channels in B-cell calcium sensing (as Ba 2ϩ is known to enter cells via calcium channels and bind Indo-1 AM) 25,52 (data not shown). However, increases in free Ca 2ϩ i concentration and CD83 expression were inhibited by tyrosine kinase and PLC␥ inhibitors (Figure 2A and data not shown), and Ca 2ϩ i release was abrogated when cells were pretreated with trypsin (which would cleave extracellular portions of cell surface receptors) or with PMA (which inhibits signaling through some cell surface receptors) [29][30][31] ( Figure 2C), suggesting that a receptor on B cells was involved in sensing Ca 2ϩ o .…”

Caffeine‐induced immobilization of gating charges in isolated guinea‐pig ventricular heart cells

“…The decrease of SL and concomitant maintained increase of the fluorescence ratio during the whole caffeine application testify of a caffeine‐induced increase of [Ca 2+ ] i , since 10 m M caffeine has been shown not to affect the Indo‐1 fluorescence ratio (O'neill et al ., 1990; McKemy et al ., 2000). Moreover, the lack of development of a contracture associated with the increase of the fluorescence ratio during application of the ‘gating’ solution alone argue against an increase of [Ca 2+ ] i induced by this solution: the best explanation for the observed increase in fluorescence is likely a modification of the Indo‐1 fluorescence emission spectrum by the high concentration of Cd 2+ used (Owen & Dever, 1995).…”

Probing Nanoelectroporation and Resealing of the Cell Membrane by the Entry of Ca2+ and Ba2+ Ions

“…Ba 2+ may also inhibit K + channels [56] and, by blocking the repolarizing action of K + channels, facilitate membrane depolarization [40]. Ba 2+ can stimulate the release of Ca 2+ from intracellular sites, block Ca 2+ extrusion from the cytosol [57,58], and modify the gating kinetics of Na + channels [59].…”