Proton-Gated Channels in PC12 Cells

Abstract

Acid-sensing ion channels (ASICs) are expressed in various sensory and central neurons. The functional role of these channels remains elusive. Complex subunit combinations and lack of specific blockers for native receptors are likely to contribute to the difficulty of resolving the function of ASICs. Finding a neuronal cell line, which expresses a single population of ASICs, should prove to be useful in delineating the function of individual ASICs. Using patch-clamp, Ca(2+)-imaging, and RT-PCR techniques, we have explored the existence of ASICs in PC12 cells, a clonal neuronal cell line. Fast drops of extracellular pH activated transient inward currents in PC12 cells with pH(0.5) at 6.0-6.2. The ASICs in PC12 cells were selective for Na(+) with significant Ca(2+) permeability. Currents in PC12 cells were blocked by the nonselective ASIC blocker amiloride. PcTX1, a specific homomeric ASIC1a blocker, also blocked the ASIC currents with an IC(50) of approximately 1.5 nM. RT-PCR demonstrated the existence of ASIC1a transcript in both undifferentiated and nerve growth factor-differentiated PC12 cells. Our data suggest that PC12 cells likely contain a single population of functional proton-gated channel-homomeric ASIC1a. It might be an ideal neuronal cell line for the study of physiological and potential pathological roles of this key subunit of ASICs.

Extracellular acidosis increases neuronal cell calcium by activating acid-sensing ion channel 1a

“…ASIC1a expressed in Xenopus oocytes has been reported to show significant Ca 2ϩ permeability (P Na ͞P Ca ϭ 2.5) (2) or a very low Ca 2ϩ permeability (P Na ͞P Ca ϭ 18.5 or greater) (23,24). Studies of isolated rat sensory neurons, PC12, and HEK-293 cells expressing endogenous ASIC1a, and COS-7 cells expressing recombinant ASIC1a have reported H ϩ -gated currents with P Na ͞P Ca values ranging from 3.2 to 50 (19)(20)(21)(22). The reason for these differences is not apparent.…”

“…It has also been proposed that ASIC1 might play a role in the pathogenesis of diseases associated with extracellular acidosis, including seizures and cerebral ischemia (18,20,41). Lactate and arachidonic acid, which are released by ischemia, might also enhance ASIC activity (13,42,43).…”

“…Previous work showed that ASICs conduct predominantly Na ϩ although other ion permeabilities have been reported. Several studies suggested that ASIC1a had an appreciable Ca 2ϩ permeability and might constitute a Ca 2ϩ entry pathway (2,(19)(20)(21)(22). Other work suggested a very low Ca 2ϩ permeability (23,24).…”

ASIC1a-Specific Modulation of Acid-Sensing Ion Channels in Mouse Cortical Neurons by Redox Reagents

“…Patch electrodes, with resistances of 2 ϳ 3 M⍀ when filled with intracellular solution, were constructed from thin-walled borosilicated glass (1.5 mm diameter; World Precision Instruments, Sarasota, FL) on a two-stage puller (PP83; Narishige, Tokyo, Japan). A multibarrel perfusion system (SF-77; Warner Instruments, Hamden, CT) was used to achieve a rapid exchange of extracellular solutions (Chu et al, 2002(Chu et al, , 2004b. Whole-cell currents or membrane potentials were recorded using Axopatch 1D amplifiers (Molecular Devices, Union City, CA).…”

“…Standard ECF contained the following (in mM): 140 NaCl, 5.4 KCl, 2.0 CaCl 2 , 1.0 MgCl 2 , 20 HEPES, and 10 glucose (pH 7.4; 320 ϳ 330 mOsm). For solutions with pH Ͻ 6.0, HEPES was replaced with MES for reliable pH buffering (Chu et al, 2002(Chu et al, , 2004b. For voltage-clamp recordings, the pipette solution contained the following (mM): 140 CsF, 10 HEPES, 11 EGTA, 2 TEA, 1 CaCl 2 , 2 MgCl 2 , and 4 K 2 ATP (pH 7.3; 300 mOsm).…”