Contribution of K_ir2 potassium channels to ATP-induced cell death in brain capillary endothelial cells and reconstructed HEK293 cell model

Abstract

]i rise induced membrane hyperpolarization via the activation of apamin-sensitive small-conductance Ca 2ϩ -activated K ϩ channels (SK2) and enhanced cell proliferation in t-BBEC 117. Here, we found anomalous membrane hyperpolarization lasting for over 10 min in response to ATP in ϳ15% of t-BBEC 117, in which inward rectifier K ϩ channel (Kir2.1) was extensively expressed. Once anomalous hyperpolarization was triggered by ATP, it was removed by Ba 2ϩ but not by apamin. Prolonged exposure to ATP␥S increased the relative population of t-BBEC 117, in which the expression of Kir2.1 mRNAs was significantly higher and Ba 2ϩ -sensitive anomalous hyperpolarization was observed. The cultivation of t-BBEC 117 in serum-free medium also increased this population and reduced the cell number. The reduction of cell number was enhanced by the addition of ATP␥S and the enhancement was antagonized by Ba 2ϩ . In the human embryonic kidney 293 cell model, where SK2 and Kir2.1 were heterologously coexpressed, [Ca 2ϩ ]i rise by P2Y stimulation triggered anomalous hyperpolarization and cell death. In conclusion, P2Y stimulation in BCECs enhances cell proliferation by SK2 activation in the majority of cells but also triggers cell death in a certain population showing a substantial expression of Kir2.1. This dual action of P2Y stimulation may effectively facilitate BCEC turnover. membrane hyperpolarization; inward rectifier K ϩ channel; Ca 2ϩ -activated K ϩ channel; human embryonic kidney 293 UNDER PHYSIOLOGICAL CONDITIONS, the blood-brain barrier (BBB) restricts the movement of substances from the circulation, blocks the invasion of noxious matter to the brain, and maintains brain homeostasis. The BBB is formed of brain capillary endothelial cells (BCECs), whose functional characteristics include the presence of intercellular tight junctions, relatively low transcellular transport, and the coordination with surrounding astrocytes (1). The robust functions of BBB require a delicate balance between the formation of new BCECs by proliferation and their elimination by cell death (13).Ion permeation through transmembrane channels regulates a variety of cell functions, not only the regulation of excitability, transmitter release, contraction, and hormone secretion in excitable cells, but also the regulation of cell volume, protein trafficking, cell metabolism, cell proliferation, and cell death even in quiescent cells, including endothelial cells (35). Apoptosis plays a critical role in embryonic development and tissue homeostasis; the balance between proliferation and apoptosis controls cell number and density. The activation of K ϩ channels has been reported as one of the major factors inducing apoptosis (24,38,54). Membrane hyperpolarization by K ϩ channel activation is supposed to increase the electrical driving force for Cl Ϫ efflux to the extracellular space and Ca 2ϩ influx though nonselective cation channels (6, 25). The cellular loss of KCl with osmotically obliged water leads to cell shrinkage, a hallmark of apoptosis (23). In add...

Membrane Hyperpolarization Induced by Endoplasmic Reticulum Stress Facilitates Ca2+ Influx to Regulate Cell Cycle Progression in Brain Capillary Endothelial Cells

“…-insensitive component of membrane hyperpolarization induced by SOCE was also blocked by 100 nM UCL1684 (normal: 0.66 ± 0.014 tunicamycin: 0.70 ± 0.019, n = 23 and 33 respectively, P = 0.17) (data not shown) and is supposed to be due to SK2 channel activation as has been suggested in the previous reports (5,8). The membrane hyperpolarization caused an increase of Kir2.1 conductance in t-BBEC117.…”

“…The membrane hyperpolarization caused an increase of Kir2.1 conductance in t-BBEC117. The excessive membrane hyperpolarization mediated by SK2 and Kir2.1 channels induced further Ca 2+ entry (5,8). These results indicated that Ca 2+ influx in ER stressloaded cells sequentially activated SK2 and Kir2.1 channels, and the subsequent membrane hyperpolarization induced further Ca 2+ entry.…”

“…We have reported previously that ATP stimulation increased [Ca 2+ ] i and triggered membrane hyperpolarization in most of t-BBEC117 under control conditions (5). The ATP-induced membrane hyperpolarization was mostly blocked by apamin, a small conductance Ca…”

“…Multiple stimuli and pathological conditions disturb ER homeostasis and result in ER stress (7) to induce damages and death of BCECs. Our previous studies indicated that the up-regulation of Kir2.1 was responsible for the elevated resting [Ca 2+ ] i and cell death in ER stress-loaded t-BBEC117 (8 ] i elevation is essential for cell proliferation/death in BCECs (5,8,11).…”

“…Whole-cell patch clamp was performed in the same way as described previously (8). The pipette solution for whole-cell patch clamp and the bath solution for the [Ca 2+ ] i and membrane potential measurement were described previously (5,8).…”