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On the excitation of action potentials by protons and its potential implications for cholinergic transmission

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Abstract

One of the most conserved mechanisms for transmission of a nerve pulse across a synapse relies on acetylcholine (ACh). Ever since the Nobel Prize-winning works of Dale and Loewi, it has been assumed that ACh—subsequent to its action on a postsynaptic cell—is split into inactive by-products by acetylcholinesterase (AChE). Herein, the widespread assumption of inactivity of ACh’s hydrolysis products is falsified. Excitable cells (Chara braunii internodes), which had previously been unresponsive to ACh, became ACh-sensitive in the presence of AChE. The latter was evidenced by a striking difference in cell membrane depolarization upon exposure to 10 mM intact ACh (∆V = −2 ± 5 mV) and its hydrolysate (∆V = 81 ± 19 mV), respectively, for 60 s. This pronounced depolarization, which also triggered action potentials, was clearly attributed to one of the hydrolysis products: acetic acid (∆V = 87 ± 9 mV at pH 4.0; choline ineffective in the range 1–10 mM). In agreement with our findings, numerous studies in the literature have reported that acids excite gels, lipid membranes, plant cells, erythrocytes, as well as neurons. Whether excitation of the postsynaptic cell in a cholinergic synapse is due to protons or due to intact ACh is a most fundamental question that has not been addressed so far.

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Notes

  1. Throughout the manuscript, the term “proton” will be used synonymously for its hydrated forms, e.g., H3O+, H5O2 +.

  2. from (Changeux and Edelstein 2005): “Initial attempts to identify the acetylcholine-binding site were hindered [because several effectors on electroplaque] also bind to […] molecules distinct from the receptor and/or have high partition coefficients in lipidic compartiments”

  3. from Matthews-Bellinger and Salpeter (1978): “We have found that [α-bungarotoxin] has a high non-specific affinity for many substrates, especially glass and some plastics including Teflon.”

  4. The following modus ponens is an over-exaggerated example: If α-bungarotoxin binds with high affinity, the acetylcholine receptor is present. α-Bungarotoxin binds to Teflon. Thus, the acetylcholine receptor must be present on Teflon.

  5. It is not possible to apply pure ACh since the compound is hydrolyzed spontaneously, yet at a slow rate, in aqueous solution.

  6. It is probable that the excitatory potency of an acid varies based on, e.g., its pK, solubility, etc.

  7. The interested reader is referred to evidence in Wunderlich et al. (2009) and Heimburg (2010) that contradicts the concept of ion translocation through protein channels.

  8. Area of endplate, ∼7000 μm2; volume of endplate, ∼450 μm3; catalytic site density, ∼2500 sites μm−2; hydrolysis rate, 0.3 molecules catalytic site−1 msec−1; ACh release, 3°106 molecules pulse−1 endplate−1 (the latter value from Potter (1970) has been quoted frequently despite the fact that the calculation and assumptions taken were not detailed in the paper). Moreover, it is not clear if residual AChE activity (after eserin treatment) was accounted for. Generously assuming 99 % of inhibition, this still leaves a hydrolysis rate of ∼5°104 molecules msec−1 endplate−1. In Potter (1970), ACh release was determined in the bath ∼300 s after repetitive stimulation. During this timespan, ∼1.5°1010 additional molecules of ACh could have been hydrolyzed per endplate and would not appear in a detection essay. Thus, the actual concentration of ACh per pulse per endplate could easily have been 3°107 molecules—an order of magnitude larger than assumed—or even higher (as argued by some, 0.1–1 mM; see discussion in Ehrenpreis (1967)). Hydrolysis of ∼107 ACh molecules will, thus, take ∼1–2 msec under physiological conditions. In the presence of cholinesterase inhibitors, the reaction is not stopped but simply extended in time to ∼10–100 msec. These order of magnitude estimates are in good agreement with experimentally obtained timescales of excitatory postsynaptic potentials in the absence and presence of anticholinesterase (compare Fig. 6 in Katz (1962)).

  9. from I. Newton’s Rules of Reasoning in Philosophy: “We are to admit no more causes of natural things than such as are both true and sufficient to explain their appearances. Therefore, to the same natural effects we must, so far as possible, assign the same causes.”

  10. For instance, butyrylcholinesterase or spontaneous hydrolysis could contribute to the liberation of protons from ACh.

  11. In fact, any variation of a thermodynamic variable (temperature; dissolution of, e.g., ethanol; change of ion concentrations; mechanical extension; etc.) to which the system is susceptible

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Acknowledgments

We thank K. Kaufmann for advocating the importance of interfaces and enzymatic “proton pistols” in biology. Moreover, we thank him for stimulating lectures and discussions. I. Silman, B. Fichtl, S. Shrivastava, H. Kong, and W. Hanke have provided helpful criticism of the manuscript. CF is grateful for funding by the Max Kade Foundation (http://maxkadefoundation.org/) and the Austrian Academy of Sciences (www.oeaw.ac.at/). MFS would like to acknowledge financial support by BU-ENG-ME and by the German Science Foundation (DFG; research unit SHENC (visiting professorship)). Chara cells for starting our cultures were kind gifts of W. Hanke, I. Foissner, M. Bisson, and R. Wayne. We also thank D. Campbell for crafting plexiglass chambers.

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  1. Biological Physics Group, Department of Mechanical Engineering, Boston University, 110 Cummington St., Boston, MA, 02215, USA

    Christian Fillafer & Matthias F. Schneider

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  1. Christian Fillafer
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Correspondence to Christian Fillafer.

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Fillafer, C., Schneider, M.F. On the excitation of action potentials by protons and its potential implications for cholinergic transmission. Protoplasma 253, 357–365 (2016). https://doi.org/10.1007/s00709-015-0815-4

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