Local subplasma membrane Ca2+ signals detected by a tethered Ca2+ sensor

Area of Science:

• Cell Biology
• Molecular Biology
• Physiology

Background:

• Plasma membrane (PM) microdomains and junctional sarcoplasmic/endoplasmic reticulum (jS/ER) form specialized Ca(2+) signaling complexes.
• Potential cross-talk between PM and jS/ER Ca(2+) signals within the junctional space is under investigation.

Purpose of the Study:

• To investigate Ca(2+) signal cross-talk between the PM and jS/ER.
• To determine the localization of Na(+) pump isoforms and their role in Ca(2+) signaling.

Main Methods:

• Engineered Ca(2+) sensor GCaMP2 fused to Na(+) pump alpha1 or alpha2 subunits.
• Expressed fusion proteins in primary cultured mouse brain astrocytes and arterial smooth muscle cells.
• Utilized immunocytochemistry and Ca(2+) imaging under varying cytosolic Ca(2+) conditions.

Main Results:

• Alpha2(f)GCaMP2 localized to PM domains adjacent to jS/ER, while alpha1(f)GCaMP2 was more uniformly distributed.
• Both constructs detected global Ca(2+) signals.
• Only alpha2(f)GCaMP2 detected localized Ca(2+) entry signals when cytosolic diffusion was restricted, suggesting alpha1 pumps are excluded from these microdomains.

Conclusions:

• The PM and jS/ER communicate via Ca(2+) signals confined to the junctional space.
• Alpha1 and alpha2 Na(+) pump isoforms exhibit distinct localizations within the PM.
• This study provides a method for investigating localized Ca(2+) signals in various cellular microdomains.