Abstract
Acid-sensing ion channels (ASICs) are voltage-independent Na(+) channels activated by extracellular protons. ASIC1a is expressed in neurons in mammalian brain and is implicated in long term potentiation of synaptic transmission that contributes to learning and memory. In ischemic brain injury, however, activation of this Ca(2+)-permeable channel plays a critical role in acidosis-mediated, glutamate-independent, Ca(2+) toxicity. We report here the identification of insulin as a regulator of ASIC1a surface expression. In modeled ischemia using Chinese hamster ovary cells, serum depletion caused a significant increase in ASIC1a surface expression that resulted in the potentiation of ASIC1a activity. Among the components of serum, insulin was identified as the key factor that maintains a low level of ASIC1a on the plasma membrane. Neurons subjected to insulin depletion increased surface expression of ASIC1a with resultant potentiation of ASIC1a currents. Intracellularly, ASIC1a is predominantly localized to the endoplasmic reticulum in Chinese hamster ovary cells, and this intracellular localization is also observed in neurons. Under conditions of serum or insulin depletion, the intracellular ASIC1a is translocated to the cell surface, increasing the surface expression level. These results reveal an important trafficking mechanism of ASIC1a that is relevant to both the normal physiology and the pathological activity of this channel.
Highlights
Health and Sciences University, 3181 SW Sam Jackson Park Rd., Portland, OR 97232
Because ASIC1a is predominantly localized in the ER in CHO cells, which is the source of surface-expressed channels in the absence of insulin, we examined the possibility of intracellular localization of ASIC1a in neuronal cells
Tight control of ASIC1a activity is vital for normal physiological function of the brain, because the synaptic activity of this channel plays a crucial role in the formation of learning and memory (3, 5)
Summary
Cell Culture, and Transfection—The rat cDNA clone of ASIC1a and ASIC2a in pCDNA3, and GFP fusion to both ASIC1a and ASIC2a at the C terminus (pCDNA-ASIC1a-EGFP and pCDNA-ASIC2a-EGFP) were described previously (13). CHO cells were cultured routinely in Dulbecco’s modified Eagle’s medium with high glucose (Invitrogen) supplemented with 10% fetal bovine serum in humidified 5% CO2 incubator. Electrophysiology—ASIC currents were recorded with whole cell patch-clamp method from stably transfected CHO cells with pCDNA-ASIC1a-EGFP and primary culture of cortical neurons from mice. The normal extracel- the surface levels of ASIC1a were insignificant when cells were lular solution contained 140 mM NaCl, 5.4 mM KCl, 2 mM treated only to low pH (pH 6) in serum- and glucose-containing. Western blot analyhad a resistance of 2– 4 M⍀ when filled with the intracellular sis of the surface-expressed ASIC1a showed that depletion of solution, which contained 140 mM CsF, 2 mM TEACl, 5 mM serum alone was sufficient to increase the expression level of EGTA, 10 mM HEPES, 1 mM CaCl2, 4 mM MgCl2 in pH 7.3 ASIC1a on the cell surface (Fig. 1C). ASIC2a is an isoform of icant when p Ͻ 0.05
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