Domains of high Ca2+ beneath the plasma membrane of living A7r5 cells

A chimera of SNAP-25 and aequorin (pmAEQ), built by replacing the SNAP-25 stop codon with a ClaI site and fusing the coding sequence in frame to HA1-tagged aequorin cDNA, localizes to the periphery of rat aortic smooth muscle A7r5 cells in confocal immunofluorescence with the anti-HA1 antibody 12CA5, with the most intense labelling in fine digitations. Cytosolic aequorin (cytAEQ) instead labels the whole cytoplasm with partial nuclear exclusion. After permeabilization with 100 μM digitonin for 2 min, plasma membrane staining persists for pmAEQ and is absent for cytAEQ.

SNAP-25 is synthesized on free ribosomes and is recruited to the inner surface of the plasma membrane after palmitoylation of specific cysteine residues, which makes it a targeting domain for delivering aequorin to the cytoplasmic rim beneath the plasma membrane. The Asp119→Ala aequorin mutant has a lower Ca2+ affinity (about 100 μM) than wild-type aequorin (about 10 μM). Fusing SNAP-25 to wild-type aequorin gives the chimera pmAEQwt, and fusing it to Asp119→Ala aequorin gives pmAEQmut, which allows [Ca2+] measurements up to 150 μM.

Reconstitution of SNAP-25–aequorin chimeras with coelenterazine (5 μM, 45 min) is strongly reduced in medium containing physiological Ca2+ (DMEM plus 1% FCS) relative to Ca2+-free Krebs–Ringer buffer, whereas cytosolic aequorin reconstitutes almost fully. Reconstituted yield in A7r5 cells is 11 ± 1% for pmAEQwt, 21 ± 4% for pmAEQmut and 88 ± 2% for cytAEQ. Because reconstitution competes with aequorin consumption by Ca2+, this pattern indirectly indicates that [Ca2+] beneath the plasma membrane is, in unstimulated cells, much higher than in the rest of the cytoplasm.

Readdition of 1 mM CaCl2 to A7r5 cells held in Ca2+-free medium (0.1 mM EGTA) causes only a marginal rise in bulk cytosolic [Ca2+] reported by cytAEQ, from a resting 0.2 μM. The same step consumes most of the pmAEQwt and pmAEQmut pool within about 2 min, and the calibrated pmAEQmut signal peaks at 120 μM, while pmAEQwt gives a calibrated peak of about 8 μM. The photon emission rates of pmAEQwt and pmAEQmut are quite similar under these conditions, which suggests that both probes are saturated, so 120 μM is only a lower limit of the real peak [Ca2+] beneath the plasma membrane and the pmAEQwt value is largely artefactual.

Readdition of 100 μM CaCl2 to A7r5 cells held in Ca2+-free medium, which changes bulk cytosolic [Ca2+] negligibly, consumes 60–70% of pmAEQwt but only 10–20% of pmAEQmut within 2 min. The calibrated [Ca2+] beneath the plasma membrane is similar for the two probes in kinetics and peak amplitude, at 8 ± 2 μM (n = 5) for pmAEQwt and 10 ± 1.5 μM (n = 5) for pmAEQmut. With pmAEQmut, [Ca2+] then settles at a steady state of 2.0 ± 0.5 μM within 2 min, significantly above basal cytosolic [Ca2+], and stays there until Ca2+ is removed from the medium.

Stimulating A7r5 cells in Ca2+-free medium with the IP3-generating agonist vasopressin (0.2 μM) raises [Ca2+] beneath the plasma membrane (pmAEQmut) with amplitude and kinetics quite similar to the bulk cytosolic rise (cytAEQ, peak 1.4 μM). This indicates that the Ca2+ affinity of aequorin is not altered by its fusion to SNAP-25 or by the local microenvironment.

Readdition of 100 μM CaCl2 to A7r5 cells whose Ca2+ stores were first emptied with vasopressin in Ca2+-free medium (store-operated Ca2+ influx) raises bulk cytosolic [Ca2+] to 0.8 μM with a sustained plateau of about 0.4 μM. The same step produces a very large and rapid rise of [Ca2+] beneath the plasma membrane, estimated at 40 ± 9 μM (n = 5) with pmAEQmut, close to saturation for pmAEQwt. Adding EGTA returns subplasmalemmal [Ca2+] to a level indistinguishable from bulk cytosolic [Ca2+].

When vasopressin (0.2 μM) is applied to A7r5 cells already in medium with 100 μM CaCl2, bulk cytosolic [Ca2+] (cytAEQ) rises transiently to a peak of 1.2 μM. Subplasmalemmal [Ca2+] (pmAEQmut) rises in the same condition from a plateau of 2 μM to about 5 μM, a larger rise than in bulk cytosol but much smaller than the peak elicited by Ca2+ readdition to store-depleted cells, probably reflecting the known inhibitory effect of Ca2+ on the store-dependent Ca2+ current.

The photon emission rate of aequorin is proportional to the 2nd–3rd power of [Ca2+], so the signal of a plasma membrane-targeted aequorin averaged over a cell is dominated by the regions of highest [Ca2+]. Maintaining a steady-state subplasmalemmal [Ca2+] above the bulk cytosol implies continuous Ca2+ flux into the cytosol, yet resting cells show no net Ca2+ flux across the plasma membrane. Transient hotspots near Ca2+ channels during spontaneous activity, with local Ca2+ extrusion keeping the net flux at zero, could account for both observations.