Electrostatic tuning of cellular excitability

Abstract

Voltage-gated ion channels regulate the electric activity of excitable tissues, such as the heart and brain. Therefore, treatment for conditions of disturbed excitability is often based on drugs that target ion channels. In this study of a voltage-gated K channel, we propose what we believe to be a novel pharmacological mechanism for how to regulate channel activity. Charged lipophilic substances can tune channel opening, and consequently excitability, by an electrostatic interaction with the channel's voltage sensors. The direction of the effect depends on the charge of the substance. This was shown by three compounds sharing an arachidonyl backbone but bearing different charge: arachidonic acid, methyl arachidonate, and arachidonyl amine. Computer simulations of membrane excitability showed that small changes in the voltage dependence of Na and K channels have prominent impact on excitability and the tendency for repetitive firing. For instance, a shift in the voltage dependence of a K channel with -5 or +5 mV corresponds to a threefold increase or decrease in K channel density, respectively. We suggest that electrostatic tuning of ion channel activity constitutes a novel and powerful pharmacological approach with which to affect cellular excitability.

Figure 1

Theory behind electrostatic tuning of voltage-sensor movement. Exemplified by three compounds with an arachidonyl backbone. (A) A negatively charged compound attracts the voltage sensor and facilitates channel opening, an uncharged substance does not affect the channel, and a positively charged substance repels the voltage sensor and hinders channel opening. The structure of arachidonic acid and its two analogs are shown to the right. (B) Schematic prediction of the effect on the voltage dependence for each compound. (C) Synthesis scheme for arachidonyl amine. Arachidonyl amine (1) was synthesized from arachidonyl alcohol (2) via arachidonyl azide (3). For the conversion from arachidonyl alcohol to arachidonyl azide (a) pyridine and methanesulphonyl chloride, followed by DMF and NaN₃ are used. For conversion from arachidonyl azide to arachidonyl amine (b) LiAlH₄, tetrahydrofuran and diethyl ether are used.

Figure 2

Effects of arachidonyl amine on the Shaker K channel. (A–C) Currents associated with voltage-clamp steps between −80 and +50 mV in 5 mV increments. Holding voltage is −80 mV. Arachidonyl amine (70 μM) reduced the steady-state current at the most negative voltages (compare black traces in B with A) and induced inactivation at more positive voltages (gray traces in B). Recovery after wash with control solution. Thick trace in A–C marks the current at −25 mV. (D–F) Currents at −50, −45, and −40 mV with (gray) and without (black) 70 μM arachidonyl amine. (G) Arachidonyl amine (70 μM) shifted the G(V) curve in positive direction along the voltage axis. Control (○), 70 μM arachidonyl amine (□), inactivation-corrected (see Materials and Methods) effect of arachidonyl (small □), inactivation-corrected recovery (small ○). Dashed curve is control curve shifted +2.3 mV. (H) Arachidonic acid-me (70 μM) did not shift the G(V) curve. Control (○), 70 μM arachidonic acid-me (□). (I) Summary of the induced G(V) shifts for different forms of arachidonyl compounds (70 μM). n = 5–8. Error bars = SE.

Figure 3

pH dependence of the arachidonyl amine effect. (A-C) Arachidonyl amine (21 μM) induced dramatic current reduction at the most negative voltages at pH 6.5. Control (black), amine (gray). (D) The G(V) shift was more pronounced at pH 6.5 than at pH 7.4. Control (○), 21 μM arachidonyl amine (□), 21 μM arachidonyl amine compensated for inactivation (small □). Dashed line is control curve shifted +5.0 mV. (E) The G(V) shift was abolished at pH 9.0 whereas the inactivation remained (see inset). Control (○), 70 μM arachidonyl amine (□), 70 μM arachidonyl amine compensated for inactivation (small □). (F) Summary of the pH dependence of arachidonyl amine effects. Error bars = SE.

Figure 4

Impact of altered voltage dependence or density of K channels on cellular excitability. Tested by computer simulations on the myelinated frog axon. (A) A persistent stimulus current of 0.6 nA (upper row) elicited one action potential in the control case. A +5 mV shift in the K channels' voltage dependence or a reduced number of K channels induced repetitive firing. A stimulus current of 1.0 nA (lower row) induced repetitive firing in the control case. A −5 mV shift in the K channels' voltage dependence or an increased number of K channels abolished repetitive firing. (B) The thick continuous line encircles the area in which repetitive firing occurs. A shift of −5 mV reduced the area of repetitive firing (small thin circle) to 0.35 of its original value (small dotted circle). A shift of +5 mV increased the area of repetitive firing (large thin circle) with a factor of 3.3 (large dotted circle). (C) The equivalent change in K channel density for different G(V) shifts. The continuous line is A = exp(−ΔV/s), where s = 4.7 mV.

Figure 5

Importance of the voltage dependencies of K and Na channels for repetitive firing tested by computer simulations of the myelinated frog axon. (A) A shift of the voltage dependence of the K channel activation in positive direction along the voltage axis decreased the threshold for repetitive firing. A shift in negative direction increased the threshold, and a shift of −2 mV abolished repetitive firing (seen as a vertical line). (B) A shift in the voltage dependence of the Na channel inactivation had similar effects as for K channel activation; a shift in positive direction reduced the threshold for repetitive firing and a shift in negative direction increased the threshold; −1 mV abolished repetitive firing. A shift of Na channel activation had the opposite effect; +1 mV abolished repetitive firing. The continuous line shows the effect of a combined shift of Na channel activation and inactivation.