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

doi:10.1073/pnas.0605757103

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

Departments of *Physiology and
^‖Medicine, University of Maryland School of Medicine, Baltimore, MD 21201;
^‡Laboratory for Memory and Learning, RIKEN Brain Science Institute, Hirosawa, Wako, Saitama 351-0198, Japan;
^§First Department of Pharmacology, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, Yoshino, Nobeoka, Miyazaki 882-8508, Japan; and
^¶Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14835

Communicated by Joseph F. Hoffman, Yale University School of Medicine, New Haven, CT, July 10, 2006 (received for review March 28, 2006)

Abstract

Accumulating evidence indicates that plasma membrane (PM) microdomains and the subjacent “junctional” sarcoplasmic/endoplasmic reticulum (jS/ER) constitute specialized Ca²⁺ signaling complexes in many cell types. We examined the possibility that some Ca²⁺ signals arising in the junctional space between the PM and jS/ER may represent cross-talk between the PM and jS/ER. The Ca²⁺ sensor protein, GCaMP2, was targeted to different PM domains by constructing genes for fusion proteins with either the α1 or α2 isoform of the Na⁺ pump catalytic (α) subunit. These fusion proteins were expressed in primary cultured mouse brain astrocytes and arterial smooth muscle cells. Immunocytochemistry demonstrated that α2(f)GCaMP2, like native Na⁺ pumps with α2-subunits, sorted to PM domains that colocalized with subjacent S/ER; α1(f)GCaMP2, like Na⁺ pumps with α1-subunits, was more uniformly distributed. The GCaMP2 moieties in both constructs were tethered just beneath the PM. Both constructs detected global Ca²⁺ signals evoked by serotonin (in arterial smooth muscle cells) and ATP, and by store-operated Ca²⁺ channel-mediated Ca²⁺ entry after S/ER unloading with cyclopiazonic acid (in Ca²⁺-free medium). When cytosolic Ca²⁺ diffusion was markedly restricted with EGTA, however, only α2(f)GCaMP2 detected the local, store-operated Ca²⁺ channel-mediated Ca²⁺ entry signal. Thus, α1 Na⁺ pumps are apparently excluded from the PM microdomains occupied by α2 Na²⁺ pumps. The jS/ER and adjacent PM may communicate by Ca²⁺ signals that are confined to the tiny junctional space between the two membranes. Similar methods may be useful for studying localized Ca²⁺ signals in other subPM microdomains and signals associated with other organelles.

Footnotes

**To whom correspondence should be addressed at:
Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201.
E-mail: mblaustein{at}som.umaryland.edu
↵ ^†Present address: Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322.
Author contributions: M.Y.L. and M.P.B. designed research; M.Y.L., H.S., and S.P.K. performed research; J.N., M.O., and M.I.K. contributed new reagents/analytic tools; V.A.G. assisted with methodology; M.Y.L. and V.A.G. analyzed data; and M.Y.L. and M.P.B. wrote the paper.
Conflict of interest statement: No conflicts declared.
Abbreviations::

ASMC,

arterial smooth muscle cell;

CPA,

cyclopiazonic acid;

5HT,

serotonin;

JS,

junctional space;

PM,

plasma membrane;

PSS,

physiological salt solution;

S/ER,

sarcoplasmic/endoplasmic reticulum;

jS/ER,

junctional S/ER;

SOC,

store-operated Ca2+ channel;

SOCE,

SOC-mediated Ca2+ entry;

DiOC,

3,3′-dihexyloxacarbocyanine iodide;

TM,

transmembrane;

AM,

membrane-permeable acetoxymethyl ester.