Dynamics of Calcium Clearance in Mouse Pancreatic β-Cells

Abstract

Pancreatic β-cells maintain glucose homeostasis by their regulated Ca²⁺-dependent secretion of insulin. Several cellular mechanisms control intracellular Ca²⁺ levels, but their relative significance in mouse β-cells is not fully known. We used photometry to measure the dynamics of cytosolic Ca²⁺ ([Ca²⁺]_i) clearance after brief, depolarization-induced Ca²⁺ entry. Treatment with thapsigargin or cyclopiazonic acid, inhibitors of the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pumps, nearly doubled the peak and slowed the decay of the depolarization-induced Ca²⁺ transients. The remaining thapsigargin-insensitive decay was slowed further by inhibition of the plasma membrane Ca²⁺-ATPase (PMCA) and plasma membrane Na⁺/Ca²⁺ exchanger (NCX) via alkalization of the bath solution, by adding lanthanum, or by substitution of Na⁺ with Li⁺. Mitochondrial Ca²⁺ uptake contributed little to clearance in thapsigargin-pretreated cells. Together, the SERCA, PMCA, and NCX transport mechanisms accounted for 89 to 97% of clearance in normal solutions. We developed a quantitative model for the dynamic role of removal mechanisms over a wide range of [Ca²⁺]_i. According to our model, 50 to 64% of initial Ca²⁺ removal is via the SERCA pump, whereas the NCX contributes 21–30% of the extrusion at high [Ca²⁺]_i, and the PMCA contributes 21–27% at low [Ca²⁺]_i.