Reversible Ca Gradients between the Subplasmalemma and Cytosol Differentially Activate Ca-dependent Cl Currents

Abstract

Xenopus oocytes express several different Ca-activated Cl currents that have different waveforms and biophysical properties. We compared the stimulation of Ca-activated Cl currents measured by two-microelectrode voltage clamp with the Ca transients measured in the same cell by confocal microscopy and Ca-sensitive fluorophores. The purpose was to determine how the amplitude and/or spatio-temporal features of the Ca signal might explain how these different Cl currents were activated by Ca. Because Ca release from stores was voltage independent, whereas Ca influx depended upon the electrochemical driving force, we were able to separately assess the contribution of Ca from these two sources. We were surprised to find that Ca signals measured with a cytosolic Ca-sensitive dye, dextran-conjugated Ca-green-1, correlated poorly with Cl currents. This suggested that Cl channels located at the plasma membrane and the Ca-sensitive dye located in the bulk cytosol were sensing different [Ca]. This was true despite Ca measurement in a confocal slice very close to the plasma membrane. In contrast, a membrane-targeted Ca-sensitive dye (Ca-green-C18) reported a Ca signal that correlated much more closely with the Cl currents. We hypothesize that very local, transient, reversible Ca gradients develop between the subplasmalemmal space and the bulk cytosol. [Ca] is higher near the plasma membrane when Ca is provided by Ca influx, whereas the gradient is reversed when Ca is released from stores, because Ca efflux across the plasma membrane is faster than diffusion of Ca from the bulk cytosol to the subplasmalemmal space. Because dissipation of the gradients is accelerated by inhibition of Ca sequestration into the endoplasmic reticulum with thapsigargin, we conclude that [Ca] in the bulk cytosol declines slowly partly due to futile recycling of Ca through the endoplasmic reticulum.

Mid-range Ca2+ signalling mediated by functional coupling between store-operated Ca2+ entry and IP3-dependent Ca2+ release

“…They faithfully report the temporal and amplitude changes in Ca 2 þ dynamics as previously described [40][41][42] . Hence, CaCCs as endogenous physiological markers of Ca 2 þ transients are superior to imaging approaches using Ca 2 þ -sensitive dyes because they remove Ca 2 þ buffering effects inherent to Ca 2 þ dyes.…”

“…CaCCs in the Xenopus oocyte provide an endogenous, sensitive and versatile reporter of Ca 2 þ transients below the PM 40 . They faithfully report the temporal and amplitude changes in Ca 2 þ dynamics as previously described [40][41][42] .…”

Orai1 internalization and STIM1 clustering inhibition modulate SOCE inactivation during meiosis

“…The exocytosis block during maturation, while endocytosis continues, leads to enrichment of channels and transporters within intracellular vesicular compartments, which are eventually inserted into the basolateral membrane of newly forming blastomeres leading to the formation of the first polarized epithelium during embryogenesis (32). GFPOrai1 distribution is not polarized in Xenopus oocytes as it is observed on both the animal and vegetal poles, consistent with the fact that Ca 2ϩ influx occurs on both poles (28,33).…”

“…3C, Ooc) with CT-STIM1 typically producing larger currents earlier, as it expressed significantly faster and to higher levels than STIM1 D76A . SOCE was measured in this case using the endogenous Ca 2ϩ -activated Cl current I Cl-T , which faithfully mirrors I SOC (27,28). Interestingly, SOCE mediated by both constitutively active mutants inactivates during maturation (Fig.…”

“…Measurements of the SOCE and Ca 2ϩ -activated Cl Ϫ currents were as previously described (11,28), except that I SOC was measured in 5Ca solution (in mM: 96 NaCl, 2.5 KCl, 5 CaCl 2, 10 Hepes pH7.4).…”