External Ba2+ block of the two-pore domain potassium channel TREK-1 defines conformational transition in its selectivity filter.

TREK-1 is a member of the two-pore domain potassium channel family that is known as a leak channel and plays a key role in many physiological and pathological processes. The conformational transition of the selectivity filter is considered as an effective strategy for potassium channels to control the course of potassium efflux. It is well known that TREK-1 is regulated by a large volume of extracellular and intracellular signals. However, until now, little was known about the selectivity filter gating mechanism of the channel. In this research, it was found that Ba(2+) blocked the TREK-1 channel in a concentration- and time-dependent manner. A mutagenesis analysis showed that overlapped binding of Ba(2+) at the assumed K(+) binding site 4 (S4) within the selectivity filter was responsible for the inhibitory effects on TREK-1. Then, Ba(2+) was used as a probe to explore the conformational transition in the selectivity filter of the channel. It was confirmed that collapsed conformations were induced by extracellular K(+)-free and acidification at the selectivity filters, leading to nonconductive to permeable ions. Further detailed characterization demonstrated that the two conformations presented different properties. Additionally, the N-terminal truncated isoform (ΔN41), a product derived from alternative translation initiation, was identified as a constitutively nonconductive variant. Together, these results illustrate the important role of selectivity filter gating in the regulation of TREK-1 by the extracellular K(+) and proton.

Knockdown of ASIC2a subunit aggravates injury of rat C6 glioma cells in acidosis

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Acid-sensing ion channel 1a (ASIC1a) and 2a (ASIC2a) subunits are widely expressed throughout mammalian central nervous system. Activation of Ca2+-permeable ASIC1a homomultimers is largely responsible for acidosis-mediated, glutamate receptor-independent, ischemic neuronal injury. The function of ASIC2a in brain ischemia is less known except that transient global ischemia induces ASIC2a protein expression up-regulation in neurons that survived ischemia. Acidosis is assumed to play a critical role in brain ischemia injury. In the present experiment, rat C6 neuroglioma cells were used to explore the function of ASIC2a. MTT and relative LDH release assay revealed that knockdown of ASIC2a could aggravate the acidosis-induced injury of C6 cells. Through changing extracellular Ca2+ concentration and measuring intracellular calcium fluorescence intensity, it was found that aggravated damage was due to toxic Ca2+ overload via ASICs mechanisms. The current results indicated that, different from ASIC1a, ASIC2a probably played a protective role against the injury induced by extracellular acidosis in C6 cells (AU)

Knockdown of ASIC2a subunit aggravates injury of rat C6 glioma cells in acidosis.

Acid-sensing ion channel 1a (ASIC1a) and 2a (ASIC2a) subunits are widely expressed throughout mammalian central nervous system. Activation of Ca²âº-permeable ASIC1a homomultimers is largely responsible for acidosis-mediated, glutamate receptorindependent, ischemic neuronal injury. The function of ASIC2a in brain ischemia is less known except that transient global ischemia induces ASIC2a protein expression up-regulation in neurons that survived ischemia. Acidosis is assumed to play a critical role in brain ischemia injury. In the present experiment, rat C6 neuroglioma cells were used to explore the function of ASIC2a. MTT and relative LDH release assay revealed that knockdown of ASIC2a could aggravate the acidosis-induced injury of C6 cells. Through changing extracellular Ca²âº concentration and measuring intracellular calcium fluorescence intensity, it was found that aggravated damage was due to toxic Ca²âº overload via ASICs mechanisms. The current results indicated that, different from ASIC1a, ASIC2a probably played a protective role against the injury induced by extracellular acidosis in C6 cells.