Mambalgin-3 potentiates human acid-sensing ion channel 1b under mild to moderate acidosis: Implications as an analgesic lead

Abstract

Acid-sensing ion channels (ASICs) are expressed in the nervous system, activated by acidosis, and implicated in pain pathways. Mambalgins are peptide inhibitors of ASIC1 and analgesic in rodents via inhibition of centrally expressed ASIC1a and peripheral ASIC1b. This activity has generated interest in mambalgins as potential therapeutics. However, most mechanism and structure-activity relationship work on mambalgins has focused on ASIC1a, and neglected the peripheral analgesic target ASIC1b. Here, we compare mambalgin potency and mechanism of action at heterologously expressed rat and human ASIC1 variants. Unlike the nanomolar inhibition at ASIC1a and rodent ASIC1b, we find mambalgin-3 only weakly inhibits human ASIC1b and ASIC1b/3 under severe acidosis, but potentiates currents under mild/moderate acidosis. Our data highlight the importance of understanding the activity of potential ASIC-targeting pharmaceuticals at human channels.

Keywords: acid-sensing ion channel; analgesic; mambalgin.

Fig. 1.

Production of Ma-3 and its species and subtype selectivity. (A) Recombinant Ma-3 has a vestigial N-terminal serine from protease cleavage. (B) Reversed-phase high-performance liquid chromatography (HPLC) chromatogram of crude cleavage reaction after affinity purification. (C) HPLC chromatogram showing >95% homogeneity and (D) matrix-assisted laser desorption ionization time-of-flight mass spectrometry spectrum showing the M+2H⁺ ion of recombinant Ma-3 (monoisotopic masses; observed, 6,648.8, and theoretical, 6,649.0). (E) Exemplar traces and concentration−response curves of Ma-3 at rat ASIC1a and ASIC1b. (F) Exemplar traces and (G) concentration−response curves of Ma-3 at human ASICs. Heteromers are from coinjection of two subtype messenger RNAs into a single oocyte. (H and I) Quantification of sustained current after 5 s of low-pH stimulus. Data are from 55-s Ma-3 application at pH 7.45, and channels were stimulated with pH 6 or 5 as indicated. (Scale bar: [E and H] abscissa 3 s, ordinate 600 nA.) For E, G, and H, Hill equation fits to the data with sufficient concentration ranges provide the Hill coefficient (n_H) and IC₅₀/EC₅₀ (EC₅₀, concentration resuling in 50% maximal effect).

Fig. 2.

Channel-specific Ma-3 mechanism of action explains human ASIC1b potentiation. (A−C) The pH dependence of activation (Upper, conditioning pH 7.45) and SSD (Lower, stimulating pH 6) for (A) rat ASIC1a, (B) human ASIC1a, and (C) rat ASIC1b. (D and E) The pH dependence of activation with exemplar traces for human ASIC1b when Ma-3 is applied in (D) only the pH 7.45 conditioning solution and (E) each of the varied low pH stimulus solutions. (Gray scale bar: abscissa 30 s, ordinate 500 nA.) (F and G) The pH dependence of SSD when Ma-3 is applied in the pH 7.45 conditioning solution and channels stimulated with (F) pH 5 and (G) pH 6. The pH dependence curves are fit to current (I) normalized to either the maximal control current (left y axis of I/I_{control max}; solid lines) or the maximal current observed in the presence of Ma-3 (right y axis of I/I_{Ma-3 max}; red dashed line). Where Ma-3 is present, concentrations are 3, 10, 30, and 1,000 nM for rASIC1a, hASIC1a, rASIC1b, and hASIC1b, respectively. Green shading in D, E, and G highlights pH conditions where Ma-3 potentiates hASIC1b currents. Hill equation fits to the data provide the color-matched pH₅₀ and Hill coefficient (n_H) for data normalized to maximal control currents.