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Different pH-sensitivity patterns of 30 sodium channel inhibitors suggest chemically different pools along the access pathway.

Lazar A, Lenkey N, Pesti K, Fodor L, Mike A - Front Pharmacol (2015)

Bottom Line: One-way to probe this is to modify the pH of the extracellular fluid, which alters the ratio of charged vs. uncharged forms of some compounds, thereby changing their interaction with the membrane.We recorded the pH-dependence of potency, reversibility, as well as onset/offset kinetics.Unexpectedly, however, the pH-dependence of reversibility or kinetics showed diverse patterns, not simple correlation.

View Article: PubMed Central - PubMed

Affiliation: Intensive Care Unit, University of Medicine and Pharmacy Tirgu Mures, Romania.

ABSTRACT
The major drug binding site of sodium channels is inaccessible from the extracellular side, drug molecules can only access it either from the membrane phase, or from the intracellular aqueous phase. For this reason, ligand-membrane interactions are as important determinants of inhibitor properties, as ligand-protein interactions. One-way to probe this is to modify the pH of the extracellular fluid, which alters the ratio of charged vs. uncharged forms of some compounds, thereby changing their interaction with the membrane. In this electrophysiology study we used three different pH values: 6.0, 7.3, and 8.6 to test the significance of the protonation-deprotonation equilibrium in drug access and affinity. We investigated drugs of several different indications: carbamazepine, lamotrigine, phenytoin, lidocaine, bupivacaine, mexiletine, flecainide, ranolazine, riluzole, memantine, ritanserin, tolperisone, silperisone, ambroxol, haloperidol, chlorpromazine, clozapine, fluoxetine, sertraline, paroxetine, amitriptyline, imipramine, desipramine, maprotiline, nisoxetine, mianserin, mirtazapine, venlafaxine, nefazodone, and trazodone. We recorded the pH-dependence of potency, reversibility, as well as onset/offset kinetics. As expected, we observed a strong correlation between the acidic dissociation constant (pKa) of drugs and the pH-dependence of their potency. Unexpectedly, however, the pH-dependence of reversibility or kinetics showed diverse patterns, not simple correlation. Our data are best explained by a model where drug molecules can be trapped in at least two chemically different environments: A hydrophilic trap (which may be the aqueous cavity within the inner vestibule), which favors polar and less lipophilic compounds, and a lipophilic trap (which may be the membrane phase itself, and/or lipophilic binding sites on the channel). Rescue from the hydrophilic and lipophilic traps can be promoted by alkalic and acidic extracellular pH, respectively.

No MeSH data available.


Related in: MedlinePlus

Examples for the different types of pH-dependence patterns exhibited by the compounds. Peak amplitudes are plotted against time during the whole experiment. Sodium currents were evoked by 5 Hz trains of 5 pulses, repeated in every 20 s. The pH of the perfusion medium was changed in the following order: neutral (black-gray-black)—acidic (red-pink-red)—neutral (black)—alkalic (dark-light-dark blue)—neutral (black-gray-black). Each major period consisted of 10 control trains, 10 trains during drug application (shown by light colors) and 10 trains during wash-out. Each plot shows the averaged normalized amplitudes of five individual experiments. All experiments were normalized to the amplitude of the current evoked by the first depolarization of the last train under control conditions. Thin black lines show the average of the five exponentials fit to individual curves, as described in Methods. Example traces from individual measurements are shown in the right panel. Black traces: Currents evoked by the first depolarization of the last control train [circled in (A)]. Gray traces: Currents evoked by the first depolarization of the last train during the first drug application period [circled in (A)]. Scale bars: 1 nA, 1 ms. (A–G) Examples for a member of each class from Class (A–G).
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Figure 1: Examples for the different types of pH-dependence patterns exhibited by the compounds. Peak amplitudes are plotted against time during the whole experiment. Sodium currents were evoked by 5 Hz trains of 5 pulses, repeated in every 20 s. The pH of the perfusion medium was changed in the following order: neutral (black-gray-black)—acidic (red-pink-red)—neutral (black)—alkalic (dark-light-dark blue)—neutral (black-gray-black). Each major period consisted of 10 control trains, 10 trains during drug application (shown by light colors) and 10 trains during wash-out. Each plot shows the averaged normalized amplitudes of five individual experiments. All experiments were normalized to the amplitude of the current evoked by the first depolarization of the last train under control conditions. Thin black lines show the average of the five exponentials fit to individual curves, as described in Methods. Example traces from individual measurements are shown in the right panel. Black traces: Currents evoked by the first depolarization of the last control train [circled in (A)]. Gray traces: Currents evoked by the first depolarization of the last train during the first drug application period [circled in (A)]. Scale bars: 1 nA, 1 ms. (A–G) Examples for a member of each class from Class (A–G).

Mentions: All electrophysiological experiments were conducted on Qpatch-16 or QPatch HT instruments (Sophion, Ballerup, Denmark) using QPlate™ chips (Kutchinsky et al., 2003; Korsgaard et al., 2009; Danker and Möller, 2014). Data were sampled at a frequency of 25 kHz and filtered at 5 kHz. Junction potential was calculated to be −11 mV and was corrected for. Histograms for the distribution of electrical properties of cells (membrane resistance, series resistance, whole-cell capacitance, current amplitude), as well as activation and steady state inactivation curves are shown in the supplement of (Lenkey et al., 2010). Cells having membrane resistance < 500 MOhm, series resistance > 9 MOhm, or capacitance > 22 pF were excluded from analysis. The experimental protocol was also identical to the one used in our previous study: sodium channels were activated by 5 Hz trains of 5 depolarizations (−90 to −10 mV) repeated in every 20 s. In this study, however, the same control–drug-application–washout sequence was repeated four times: at neutral, acidic, alkalic, and again neutral pH. The peak amplitudes of sodium currents throughout the whole experiment are shown plotted against time in Figure 1 and Supplementary Figure 1.


Different pH-sensitivity patterns of 30 sodium channel inhibitors suggest chemically different pools along the access pathway.

Lazar A, Lenkey N, Pesti K, Fodor L, Mike A - Front Pharmacol (2015)

Examples for the different types of pH-dependence patterns exhibited by the compounds. Peak amplitudes are plotted against time during the whole experiment. Sodium currents were evoked by 5 Hz trains of 5 pulses, repeated in every 20 s. The pH of the perfusion medium was changed in the following order: neutral (black-gray-black)—acidic (red-pink-red)—neutral (black)—alkalic (dark-light-dark blue)—neutral (black-gray-black). Each major period consisted of 10 control trains, 10 trains during drug application (shown by light colors) and 10 trains during wash-out. Each plot shows the averaged normalized amplitudes of five individual experiments. All experiments were normalized to the amplitude of the current evoked by the first depolarization of the last train under control conditions. Thin black lines show the average of the five exponentials fit to individual curves, as described in Methods. Example traces from individual measurements are shown in the right panel. Black traces: Currents evoked by the first depolarization of the last control train [circled in (A)]. Gray traces: Currents evoked by the first depolarization of the last train during the first drug application period [circled in (A)]. Scale bars: 1 nA, 1 ms. (A–G) Examples for a member of each class from Class (A–G).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4585259&req=5

Figure 1: Examples for the different types of pH-dependence patterns exhibited by the compounds. Peak amplitudes are plotted against time during the whole experiment. Sodium currents were evoked by 5 Hz trains of 5 pulses, repeated in every 20 s. The pH of the perfusion medium was changed in the following order: neutral (black-gray-black)—acidic (red-pink-red)—neutral (black)—alkalic (dark-light-dark blue)—neutral (black-gray-black). Each major period consisted of 10 control trains, 10 trains during drug application (shown by light colors) and 10 trains during wash-out. Each plot shows the averaged normalized amplitudes of five individual experiments. All experiments were normalized to the amplitude of the current evoked by the first depolarization of the last train under control conditions. Thin black lines show the average of the five exponentials fit to individual curves, as described in Methods. Example traces from individual measurements are shown in the right panel. Black traces: Currents evoked by the first depolarization of the last control train [circled in (A)]. Gray traces: Currents evoked by the first depolarization of the last train during the first drug application period [circled in (A)]. Scale bars: 1 nA, 1 ms. (A–G) Examples for a member of each class from Class (A–G).
Mentions: All electrophysiological experiments were conducted on Qpatch-16 or QPatch HT instruments (Sophion, Ballerup, Denmark) using QPlate™ chips (Kutchinsky et al., 2003; Korsgaard et al., 2009; Danker and Möller, 2014). Data were sampled at a frequency of 25 kHz and filtered at 5 kHz. Junction potential was calculated to be −11 mV and was corrected for. Histograms for the distribution of electrical properties of cells (membrane resistance, series resistance, whole-cell capacitance, current amplitude), as well as activation and steady state inactivation curves are shown in the supplement of (Lenkey et al., 2010). Cells having membrane resistance < 500 MOhm, series resistance > 9 MOhm, or capacitance > 22 pF were excluded from analysis. The experimental protocol was also identical to the one used in our previous study: sodium channels were activated by 5 Hz trains of 5 depolarizations (−90 to −10 mV) repeated in every 20 s. In this study, however, the same control–drug-application–washout sequence was repeated four times: at neutral, acidic, alkalic, and again neutral pH. The peak amplitudes of sodium currents throughout the whole experiment are shown plotted against time in Figure 1 and Supplementary Figure 1.

Bottom Line: One-way to probe this is to modify the pH of the extracellular fluid, which alters the ratio of charged vs. uncharged forms of some compounds, thereby changing their interaction with the membrane.We recorded the pH-dependence of potency, reversibility, as well as onset/offset kinetics.Unexpectedly, however, the pH-dependence of reversibility or kinetics showed diverse patterns, not simple correlation.

View Article: PubMed Central - PubMed

Affiliation: Intensive Care Unit, University of Medicine and Pharmacy Tirgu Mures, Romania.

ABSTRACT
The major drug binding site of sodium channels is inaccessible from the extracellular side, drug molecules can only access it either from the membrane phase, or from the intracellular aqueous phase. For this reason, ligand-membrane interactions are as important determinants of inhibitor properties, as ligand-protein interactions. One-way to probe this is to modify the pH of the extracellular fluid, which alters the ratio of charged vs. uncharged forms of some compounds, thereby changing their interaction with the membrane. In this electrophysiology study we used three different pH values: 6.0, 7.3, and 8.6 to test the significance of the protonation-deprotonation equilibrium in drug access and affinity. We investigated drugs of several different indications: carbamazepine, lamotrigine, phenytoin, lidocaine, bupivacaine, mexiletine, flecainide, ranolazine, riluzole, memantine, ritanserin, tolperisone, silperisone, ambroxol, haloperidol, chlorpromazine, clozapine, fluoxetine, sertraline, paroxetine, amitriptyline, imipramine, desipramine, maprotiline, nisoxetine, mianserin, mirtazapine, venlafaxine, nefazodone, and trazodone. We recorded the pH-dependence of potency, reversibility, as well as onset/offset kinetics. As expected, we observed a strong correlation between the acidic dissociation constant (pKa) of drugs and the pH-dependence of their potency. Unexpectedly, however, the pH-dependence of reversibility or kinetics showed diverse patterns, not simple correlation. Our data are best explained by a model where drug molecules can be trapped in at least two chemically different environments: A hydrophilic trap (which may be the aqueous cavity within the inner vestibule), which favors polar and less lipophilic compounds, and a lipophilic trap (which may be the membrane phase itself, and/or lipophilic binding sites on the channel). Rescue from the hydrophilic and lipophilic traps can be promoted by alkalic and acidic extracellular pH, respectively.

No MeSH data available.


Related in: MedlinePlus