Acidosis decreases low Ca²⁺ ‐induced neuronal excitation by inhibiting the activity of calcium‐sensing cation channels in cultured mouse hippocampal neurons

Abstract

The effects of extracellular pH (pHo) on calcium‐sensing non‐selective cation (csNSC) channels in cultured mouse hippocampal neurons were investigated using whole‐cell voltage‐clamp and current‐clamp recordings. Decreasing extracellular Ca2+ concentrations ([Ca2+]o) activated slow and sustained inward currents through the csNSC channels. Decreasing pHo activated amiloride‐sensitive transient proton‐gated currents which decayed to baseline in several seconds. With proton‐gated channels inactivated by pre‐perfusion with low pH solution or blocked by amiloride, decreasing pHo to 6.5 inhibited the csNSC currents with a leftward shift of the Ca2+ dose–inhibition curve. Increasing pH to 8.5, on the other hand, caused a rightward shift of the Ca2+ dose–inhibition curve and potentiated the csNSC currents. Intracellular alkalinization following bath perfusion of quinine mimicked the potentiation of the csNSC currents by increasing pHo, while intracellular acidification by addition and subsequent withdrawal of NH4Cl mimicked the inhibition of the csNSC currents by decreasing pHo. Intracellular pH (pHi) imaging demonstrated that decreasing pHo induced a corresponding decrease in pHi. Including 30 mM Hepes in the pipette solution eliminated the effects of quinine and NH4Cl on the csNSC currents, but only partially reduced the effect of lowering pHo. In current‐clamp recordings, decreasing [Ca2+]o induced sustained membrane depolarization and excitation of hippocampal neurons. Decreasing pHo to 6.5 inhibited the low [Ca2+]o‐induced csNSC channel‐mediated membrane depolarization and the excitation of neurons. Our results indicate that acidosis may inhibit low [Ca2+]o‐induced neuronal excitation by inhibiting the activity of the csNSC channels. Both the extracellular and the intracellular sites are involved in the proton modulation of the csNSC channels.

Modulation of Acid-sensing Ion Channel Currents, Acid-induced Increase of Intracellular Ca2+, and Acidosis-mediated Neuronal Injury by Intracellular pH

“…Primary Neuronal Culture-Primary cortical neuronal cultures were prepared from embryonic Swiss mice at 16 days of gestation according to our previously described method (10,23). In brief, the brains of embryos were rapidly removed and incubated in Ca 2ϩ -and Mg 2ϩ -free ice-cold phosphate-buffered saline.…”

“…Electrophysiology-Whole cell patch clamp recordings were performed as described previously (10,23). Patch pipettes were pulled by a two-step puller (PP83; Narishige, Tokyo, Japan) from thin wall borosilicated glass (1.5 mm diameter; WPI, Sarasota, FL).…”

“…To determine whether a change in pH i affects the ASIC current, we first applied quinine, an agent known to cause intracellular alkalosis (23,24), to the extracellular solution, following the recording of stable ASIC currents activated by a pH o drop from 7.4 to 6.5. In our previous studies, we have shown that bath perfusion of 0.5 mM quinine for 2 min increased pH i by ϳ0.5 unit (23).…”

Calcium sensitive non-selective cation current promotes seizure-like discharges and spreading depression in a model neuron

“…The main expansion of our model compared to its previous versions was the implementation of the Casensitive nonselective cation conductance, g NSCC , (based on data given in Chu et al 2003;Xiong et al 1997 and Xiong, personal communication). The nonspecific cation channel senses the extracellular calcium concentration ( Fig.…”

“…Hablitz et al (1986) suggested that a similar current in central neurons could play a role in the generation of seizures and spreading depression (SD). MacDonald, Xiong, and collaborators (Chu et al 2003;Xiong and MacDonald 1999;Xiong et al 1997) detected in mammalian central neurons an NSCC that is activated by the lowering of [Ca 2+ ] o . This channel is maximally open when extracellular calcium concentration is zero, and at the normal [Ca 2+ ] o of 1.2-1.5 mM it permits the flow of a small current which, presumably, adds to the resting conductance of the cell.…”