Clearance of store-released Ca²⁺ by the Na⁺-Ca ²⁺ exchanger is diminished in aortic smooth muscle from Na ⁺-K⁺-ATPase α₂-isoform gene-ablated mice

Two α-isoforms of the Na⁺-K⁺-ATPase are expressed in vascular smooth muscle cells (VSMCs). The α₁- isoform is proposed to serve a cytosolic housekeeping role, whereas the α₂-isoform modulates Ca²⁺ storage via coupling to the Na⁺-Ca²⁺ exchanger (NCX) in a subsarcolemmal compartment. To evaluate the ramifications of this proposed interaction, Ca ²⁺-store load and the contributions of the primary Ca²⁺ transporters to Ca²⁺ clearance were studied in aortic VSMCs from embryonic wild-type (WT) and Na⁺-K⁺-ATPase α₂-isoform gene-ablated, homozygous null knockout (α₂-KO) mice. Ca²⁺ stores were unloaded by inhibiting the sarco(endo)plasmic reticulum Ca²⁺-ATPase with cyclopiazonic acid (CPA) in Ca²⁺-free media to limit Ca²⁺ influx. Ca²⁺ clearance by the plasma membrane Ca²⁺-ATPase (PMCA), NCX, or mitochondria was selectively inhibited. In WT VSMCs, NCX accounted for 90% of the Ca²⁺ efflux. In α₂-KO VSMCs, preferential clearance of store-released Ca²⁺ by NCX was lost, whereas PMCA activity was increased. Selective inhibition of the α₂-isoform (0.5 μM ouabain for 20 min), before treatment with CPA enhanced the store load in VSMCs from WT, but not α₂- KO mice. A subsequent analysis of capacitative Ca²⁺ entry (CCE) indicated that the magnitude of Ca²⁺ influx was significantly greater in α₂-KO cells. Our findings support the concept of a subsarcolemmal space where the α₂-isoform coupled with NCX modulates Ca²⁺-store function and, thereby, CCE.