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Identifying the Ca(++) signalling sources activating chloride currents in Xenopus oocytes using ionomycin and
C L Thurman1, J S Burns, R G O'Neil
1Department of Biology, University of Northern Iowa, Cedar Falls, IA 50614-0421, USA. Thurman@uni.edu
Abstract:
The calcium ionophore, ionomycin (IM), and the sarcoplasmic/endoplasmic reticulum (SER) calcium pump inhibitor, thapsigargin (TG), were used to study the roles of Ca(++) from different sources in regulating Ca(++)-dependent Cl(-) currents in Xenopus oocytes. The Ca(++)-dependent Cl(-) currents, Ic, were measured in voltage-clamped oocytes (Vc = -60 mV). In the presence of extracellular Ca(++), both TG (0.1 to 10 microM) and IM (0.1 to 10 microM) induce release of Ca(++) from SER and activated capacitative Ca(++) entry (CCE) across the plasma membrane leading to activation of both "fast" and "slow" Cl(-) currents. The fast Ic was produced by Ca(++) release from SER while Ca(++) entry across the plasma membrane activated the slow Ic. Intracellular application of the calcium buffer, BAPTA, blocked activation of the slow Ic due to Ca(++) entry via CCE pathways, but not via IM-mediated movement across the plasma membrane. It is concluded that predominantly Ca(++) release from stores regulates a fast Ic while Ca(++) entry through CCE pathways regulates a slow Ic. Further, the CCE and slow Ic pathways must be located in spatially separated compartments since BAPTA can effectively abolish the effects of Ca(++) entry via the CCE pathway, but not by the IM-mediated entry pathway.
Insights
Calcium release from internal stores regulates fast chloride currents, while calcium entry via capacitative calcium entry (CCE) activates slow currents in Xenopus oocytes. These pathways are spatially separated.
Area of Science:
- Cellular Physiology
- Ion Channel Function
- Calcium Signaling
Background:
- Calcium ions (Ca++) are crucial intracellular messengers regulating various cellular processes.
- Calcium-dependent chloride currents (Ic) play significant roles in oocyte function.
- Understanding the sources and pathways of calcium influx is key to elucidating Ic regulation.
Purpose of the Study:
- To investigate the distinct roles of intracellular and extracellular calcium in activating calcium-dependent chloride currents (Ic) in Xenopus oocytes.
- To differentiate the mechanisms underlying fast and slow Ic activation.
- To explore the spatial localization of calcium entry pathways.
Main Methods:
- Utilized ionomycin (IM) and thapsigargin (TG) to manipulate intracellular calcium levels.
- Measured Ca(++)-dependent Cl(-) currents (Ic) in voltage-clamped Xenopus oocytes.
- Employed intracellular calcium buffering with BAPTA to assess calcium entry pathways.
Main Results:
- Both IM and TG induced Ca(++) release from the sarcoplasmic/endoplasmic reticulum (SER) and activated capacitative Ca(++) entry (CCE).
- Fast Ic was primarily driven by SER Ca(++) release, while slow Ic was activated by plasma membrane Ca(++) entry.
- BAPTA blocked slow Ic from CCE but not IM-mediated entry, indicating distinct spatial compartments.
Conclusions:
- Spatially distinct mechanisms regulate fast and slow calcium-dependent chloride currents.
- Intracellular Ca(++) stores primarily control fast Ic, whereas CCE pathways govern slow Ic.
- IM and CCE pathways operate in separate cellular compartments, as evidenced by differential BAPTA sensitivity.
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