Endoplasmic reticulum potassium-hydrogen exchanger and small conductance calcium-activated potassium channel activities are essential for ER calcium uptake in neurons and cardiomyocytes

Calcium pumping into the endoplasmic reticulum (ER) lumen is thought to be coupled to a countertransport of protons through sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) and the members of the ClC family of chloride channels. However, pH in the ER lumen remains neutral, which suggests a mechanism responsible for proton re-entry. We studied whether cation proton exchangers could act as routes for such a re-entry. ER Ca2+ uptake was measured in permeabilized immortalized hypothalamic neurons, primary rat cortical neurons and mouse cardiac fibers. Replacement of K+ in the uptake solution with Na+ or tetraethylammonium led to a strong inhibition of Ca2+ uptake in neurons and cardiomyocytes. Furthermore, inhibitors of the potassium proton exchanger (quinine or propranolol) but not of the sodium proton exchanger reduced ER Ca2+ uptake by 56-82%. Externally added nigericin, a potassium proton exchanger, attenuated the inhibitory effect of propranolol. Inhibitors of small conductance calcium-sensitive K+ (SKCa) channels (UCL 1684, dequalinium) blocked the uptake of Ca2+ by the ER in all preparations by 48-94%, whereas inhibitors of other K+ channels (IKCa, BKCa and K-ATP) had no effect. Fluorescence microscopy and western blot analysis revealed the presence of both SKCa channels and the potassium proton exchanger leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1) in ER in situ and in the purified ER fraction. The data obtained demonstrate that SKCa channels and LETM1 reside in the ER membrane and that their activity is essential for ER Ca2+ uptake.