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Measuring Endogenous $I_{CRAC}$ and ORAI Currents with the Patch-Clamp Technique

The Minimal Requirements to Use Calcium Imaging to Analyze $I_{CRAC}$

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Patch-Clamp Measurement of $I_{CRAC}$ and ORAI Channel Activity

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The Minimal Requirements to Use Calcium Imaging to Analyze $I_{CRAC}$

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Alansary, Dalia Kilch, Tatiana Holzmann, Christian Peinelt, Christine Hoth, Markus Lis, Annette
Description	Country affiliation: Germany Author Affiliation: Alansary D ( Department of Biophysics, Saarland University, Homburg, Germany.); Kilch T ( Department of Biophysics, Saarland University, Homburg, Germany.); Holzmann C ( Department of Biophysics, Saarland University, Homburg, Germany.); Peinelt C ( Department of Biophysics, Saarland University, Homburg, Germany.); Hoth M ( Department of Biophysics, Saarland University, Homburg, Germany.); Lis A ( Department of Biophysics, Saarland University, Homburg, Germany.)
Abstract	Endogenous calcium release-activated channel (CRAC) currents are usually quite small and not always easy to measure using the patch-clamp technique. While we have, for instance, successfully recorded very small CRAC currents in primary human effector T cells, we have not yet managed to record CRAC in naïve primary human T cells. Many groups, including ours, therefore use $Ca^{2+}$ imaging technologies to analyze CRAC-dependent $Ca^{2+}$ influx. However, $Ca^{2+}$ signals are quite complex and depend on many different transporter activities; thus, it is not trivial to make quantitative statements about one single transporter, in this case CRAC channels. Therefore, a detailed patch-clamp analysis of $I_{CRAC}$ is always preferred. Since many laboratories use $Ca^{2+}$ imaging for $I_{CRAC}$ analysis, we detail here the minimal requirements for reliable measurements. $Ca^{2+}$ signals not only depend on the net $Ca^{2+}$ influx through CRAC channels but also depend on other $Ca^{2+}$ influx mechanisms, $K^{+}$ channels or $Cl^{−}$ channels (which determine the membrane potential), $Ca^{2+}$ export mechanisms like plasma membrane $Ca^{2+}$ ATPase (PMCA), sarco/endoplasmic reticulum $Ca^{2+}$ ATPase (SERCA) or $Na^{+}–Ca^{2+}$ exchangers, and (local) $Ca^{2+}$ buffering often by mitochondria. In this protocol, we summarize a set of experiments that allow (quantitative) statements about CRAC channel activity using $Ca^{2+}$ imaging experiments, including the ability to rule out $Ca^{2+}$ signals from other sources.
File Format	HTM / HTML
ISSN	19403402
Issue Number	6
Volume Number	2014
e-ISSN	15596095
Journal	Cold Spring Harbor Protocols
Language	English
Publisher	Cold Spring Harbor Laboratory Press
Publisher Date	2014-06-02
Publisher Place	United States
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Discipline Clinical Laboratory Techniques Calcium Channels Metabolism Calcium Optical Imaging Methods Animals Cells, Cultured Humans Journal Article Research Support, Non-u.s. Gov't
Content Type	Text
Resource Type	Article
Subject	Biochemistry, Genetics and Molecular Biology

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3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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Measuring Endogenous $I_{CRAC}$ and ORAI Currents with the Patch-Clamp Technique

Patch-Clamp Measurement of $I_{CRAC}$ and ORAI Channel Activity

Activation of calcium entry in human carcinoma A431 cells by store depletion and phospholipase C- dependent mechanisms converge on $I_{CRAC}-like$ calcium channels

Two-Dimensional Imaging of Fast Intracellular $Ca^{2+}$ Release

Patch-Clamp Recording of Voltage-Sensitive $Ca^{2+}$ Channels

Contribution of BK channels to action potential repolarisation at minimal cytosolic $Ca^{2+}$ concentration in chromaffin cells

Two-photon uncaging microscopy.

Whole-Cell Patch-Clamp Recording of Recombinant Voltage-Sensitive $Ca^{2+}$ Channels Heterologously Expressed in HEK-293 Cells

Calcium influx through hyperpolarization-activated cation channels $(I_{h}$ channels) contributes to activity-evoked neuronal secretion

The Minimal Requirements to Use Calcium Imaging to Analyze $I_{CRAC}$

Similar Documents

Measuring Endogenous $I_{CRAC}$ and ORAI Currents with the Patch-Clamp Technique

Patch-Clamp Measurement of $I_{CRAC}$ and ORAI Channel Activity

Activation of calcium entry in human carcinoma A431 cells by store depletion and phospholipase C- dependent mechanisms converge on $I_{CRAC}-like$ calcium channels

Two-Dimensional Imaging of Fast Intracellular $Ca^{2+}$ Release

Patch-Clamp Recording of Voltage-Sensitive $Ca^{2+}$ Channels

Contribution of BK channels to action potential repolarisation at minimal cytosolic $Ca^{2+}$ concentration in chromaffin cells

Two-photon uncaging microscopy.

Whole-Cell Patch-Clamp Recording of Recombinant Voltage-Sensitive $Ca^{2+}$ Channels Heterologously Expressed in HEK-293 Cells

Calcium influx through hyperpolarization-activated cation channels $(I_{h}$ channels) contributes to activity-evoked neuronal secretion

The Minimal Requirements to Use Calcium Imaging to Analyze $I_{CRAC}$