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. 1998 Aug 18;95(17):10240-5.
doi: 10.1073/pnas.95.17.10240.

A sensory neuron-specific, proton-gated ion channel

C C Chen  1 , S England, A N Akopian, J N Wood
Affiliations

A sensory neuron-specific, proton-gated ion channel

C C Chen et al. Proc Natl Acad Sci U S A. .

Abstract

Proton-gated channels expressed by sensory neurons are of particular interest because low pH causes pain. Two proton-gated channels, acid-sensing ionic channel (ASIC) and dorsal root ASIC (DRASIC), that are members of the amiloride-sensitive ENaC/Degenerin family are known to be expressed by sensory neurons. Here, we describe the cloning and characterization of an ASIC splice variant, ASIC-beta, which contains a unique N-terminal 172 aa, as well as unique 5' and 3' untranslated sequences. ASIC-beta, unlike ASIC and DRASIC, is found only in a subset of small and large diameter sensory neurons and is absent from sympathetic neurons or the central nervous system. The patterns of expression of ASIC and ASIC-beta transcripts in rat dorsal root ganglion neurons are distinct. When expressed in COS-7 cells, ASIC-beta forms a functional channel with electrophysiological properties distinct from ASIC and DRASIC. The pH dependency and sensitivity to amiloride of ASIC-beta is similar to that described for ASIC, but unlike ASIC, the channel is not permeable to calcium, nor are ASIC-beta-mediated currents inhibited by extracellular calcium. The unique distribution of ASIC-beta suggests that it may play a specialized role in sensory neuron function.

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Figures

Figure 1
Figure 1
Structure of ASIC splice variants and primary sequence alignment of ASIC-β with four other known members of the family. (A) The gene structure of ASIC splice variants. Three different transcripts are distinguished by their 5′UTRs (hatched) but share the same 3′UTR (white). The coding regions are black apart from the unique N terminus of ASIC-β (striped). (B) The proposed molecular structures of ASIC-α and ASIC-β shows that both proteins have the same two transmembrane domain structure, with intracellular N and C terminals. The striped region of AIC-β shows its unique N-terminal region including the first transmembrane domain. (C) The N-terminal sequence alignment of four ASIC-related proteins. Block letters present the cysteine resides that are conserved amongst the four proteins, implying a similar secondary structure.
Figure 2
Figure 2
Northern blots of ASIC-β distribution. The Northern blots were probed by N-terminal unique sequences of ASIC-α, ASIC-β, and DRASIC. All three proton-gated channels are expressed in sensory neurons. ASIC-α is distributed in many neural tissues and cell lines. There are three different sizes of ASIC-α transcripts in PC12 cells that are 2.5, 3.2, and 4.0 kb, but there is only one major transcript of 3.2 kb in sensory neurons. ASIC-β is only expressed in DRG as a 3.2-kb transcript. DRASIC is predominantly in DRG with two sizes of transcripts, 2.0 and 2.5 kb, but also is expressed in superior cervical ganglion, spinal cord, and brain stem. The relative amount of RNA loading is indicated by cyclophilin probe.
Figure 3
Figure 3
Cell specificity of ASIC-α and ASIC-β in DRG. In situ hybridization of ASIC-α and ASIC-β, double-labeled with anti-peripherin (A–I) or isolectin B4 (J–O). The left column is overlayed images of in situ (middle column) and immunocytochemistry (right column) results. The expression of ASIC-α transcript is most (>90%) colocalized with peripherin (A–C, indicated by arrows). However, ASIC-β transcript is distributed in either peripherin-positive (30%) or peripherin-negative (70%) neurons (D–I). The colocalization of ASIC-β and peripherin is indicated by arrows (G and H). The expression of ASIC-β in peripherin-negative large diameter neurones is shown by arrowheads (D and E). Neither ASIC-α (M–O, indicated by arrowheads) nor ASIC-β (J–L, indicated by arrowheads) have colocalized staining with isolectin B4, which corresponds to GDNF-dependent small diameter neurons.
Figure 4
Figure 4
Characteristics of the pH response in COS-7 cells expressing ASIC-β. Typical response to low pH in ASIC-β transfected COS cells. The cell was voltage-clamped at −60 mV and low pH applied at the bar. Dotted line indicates zero current level. (B) pH response relationship obtained from experiments similar to that in A. Responses were normalized against the maximal response and plotted against the pH. The half-point for activation of the current was pH 5.9. (C) Time taken for the current to activate and inactivate plotted against pH. (D) Recordings made during a change in command potential by using a linear ramp protocol (duration of ramp 240 ms). Current was recorded under control conditions and during application of low pH. The current reverses at approximately +25 mV.
Figure 5
Figure 5
Calcium dependency and pharmacology of ASIC-β-mediated currents in COS cells. (A) Responses obtained to pH 5.1 (at the closed circles) in the presence of increased extracellular calcium concentration. Recordings were made from the same cell at intervals of 3 min. (B, Left) Control response to pH 5.1 and (Right) responses to low pH in the absence of extracellular sodium and increased calcium concentration are shown. Current flowing via ASIC-β is not inhibited by extracellular calcium, nor is the channel permeable to calcium. Dotted line indicates zero current level. (C) Amiloride inhibits ASIC-β mediated current. The IC50 derived from this plot was 21 μM. (D) Capsaicin does not activate ASIC-β. Recordings made from the same cell; holding potential was −60 mV. Upper trace shows that application of capsaicin (500 nM) at the bar failed to evoke an inward current. pH 4.1, at the bar 3 min later (lower trace), evoked a robust inward current. Traces have been separated for clarity, and the dotted line indicates zero current for each recording.
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