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Mechanism of block of hEag1 K+ channels by imipramine and astemizole.

García-Ferreiro RE, Kerschensteiner D, Major F, Monje F, Stühmer W, Pardo LA - J. Gen. Physiol. (2004)

Bottom Line: Using inside- and outside-out patch recordings, we found that a permanently charged, quaternary derivative of imipramine (N-methyl-imipramine) only blocks channels from the intracellular side of the membrane.However, as astemizole competes with imipramine and intracellular TEA for binding to the channel, it is proposed to interact with an overlapping intracellular binding site.The significance of these findings, in the context of structure-function of channels of the eag family is discussed.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Molekulare Biologie Neuronaler Signale, Max-Planck Institut für Experimentelle Medizin, 37075 Göttingen, Germany. rgarcia@gwdg.de

ABSTRACT
Ether à go-go (Eag; KV10.1) voltage-gated K+ channels have been detected in cancer cell lines of diverse origin and shown to influence their rate of proliferation. The tricyclic antidepressant imipramine and the antihistamine astemizole inhibit the current through Eag1 channels and reduce the proliferation of cancer cells. Here we describe the mechanism by which both drugs block human Eag1 (hEag1) channels. Even if both drugs differ in their affinity for hEag1 channels (IC50s are approximately 2 microM for imipramine and approximately 200 nM for astemizole) and in their blocking kinetics, both drugs permeate the membrane and inhibit the hEag1 current by selectively binding to open channels. Furthermore, both drugs are weak bases and the IC50s depend on both internal an external pH, suggesting that both substances cross the membrane in their uncharged form and act from inside the cell in their charged forms. Accordingly, the block by imipramine is voltage dependent and antagonized by intracellular TEA, consistent with imipramine binding in its charged form to a site located close to the inner end of the selectivity filter. Using inside- and outside-out patch recordings, we found that a permanently charged, quaternary derivative of imipramine (N-methyl-imipramine) only blocks channels from the intracellular side of the membrane. In contrast, the block by astemizole is voltage independent. However, as astemizole competes with imipramine and intracellular TEA for binding to the channel, it is proposed to interact with an overlapping intracellular binding site. The significance of these findings, in the context of structure-function of channels of the eag family is discussed.

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Concentration dependence of hEag1 block by imipramine and astemizole. (A and C) Superimposed hEag1 current traces recorded during 1.5 s test depolarizations to 80 mV from a holding potential of −70 mV in the absence and presence of the indicated concentrations of imipramine (Imi, A) or astemizole (Ast, C). Test potential was chosen to achieve the maximal open probability of hEag1, whose activation curve saturates above 60 mV (not depicted). The effects of drug application were monitored with test pulses applied every 30 s until a steady-state block was reached. (B and D) Current traces in the presence of imipramine or astemizole were normalized dividing them point by point by the respective preapplication traces. Solid lines indicate the best fit to a single exponential function. (E) Dose–response plots for imipramine (open circles) and astemizole (closed circles). The steady-state fraction of channels blocked was calculated from the asymptotic values of single exponential fits to current ratios as shown in B and D. Solid lines represent fits to the data using the Hill equation, with IC50 values and Hill coefficients of 1.87 μM and 1.04 for imipramine, and 0.21 μM and 1.32 for astemizole, respectively. (D) Time constant of block (τblock) for imipramine (open circles) and astemizole (closed circles) derived from the least-squares fits of single exponential functions used in E. Solid lines represent fits to the data using the Hill equations, with maximum, minimum, IC50, and Hill coefficients of 86.7 ms, 11.6 ms, 3.75 μM, and 1.27 for imipramine, and 1.33 s, 0.024 s, 0.26 μM, and 1.32 for astemizole, respectively. (G) The rate of current block is represented (τblock−1) as a linear function of nonsaturating imipramine (open circles) or astemizole (closed circles) concentrations. Solid lines represent fits to the data with a linear function, with slope and y intercept of 2.5 s−1μM−1 and 11.1 μM for imipramine, and 4 s−1μM−1 and 0.4 μM for astemizole, respectively. The range of drug concentrations used to fit τblock−1 data to the linear function was between 0.5 and 10 μM for imipramine and between 25 nM and 5 μM for astemizole. Symbols and associated error bars in E–G represent means ± SEM for six and seven cells for imipramine and astemizole, respectively.
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fig2: Concentration dependence of hEag1 block by imipramine and astemizole. (A and C) Superimposed hEag1 current traces recorded during 1.5 s test depolarizations to 80 mV from a holding potential of −70 mV in the absence and presence of the indicated concentrations of imipramine (Imi, A) or astemizole (Ast, C). Test potential was chosen to achieve the maximal open probability of hEag1, whose activation curve saturates above 60 mV (not depicted). The effects of drug application were monitored with test pulses applied every 30 s until a steady-state block was reached. (B and D) Current traces in the presence of imipramine or astemizole were normalized dividing them point by point by the respective preapplication traces. Solid lines indicate the best fit to a single exponential function. (E) Dose–response plots for imipramine (open circles) and astemizole (closed circles). The steady-state fraction of channels blocked was calculated from the asymptotic values of single exponential fits to current ratios as shown in B and D. Solid lines represent fits to the data using the Hill equation, with IC50 values and Hill coefficients of 1.87 μM and 1.04 for imipramine, and 0.21 μM and 1.32 for astemizole, respectively. (D) Time constant of block (τblock) for imipramine (open circles) and astemizole (closed circles) derived from the least-squares fits of single exponential functions used in E. Solid lines represent fits to the data using the Hill equations, with maximum, minimum, IC50, and Hill coefficients of 86.7 ms, 11.6 ms, 3.75 μM, and 1.27 for imipramine, and 1.33 s, 0.024 s, 0.26 μM, and 1.32 for astemizole, respectively. (G) The rate of current block is represented (τblock−1) as a linear function of nonsaturating imipramine (open circles) or astemizole (closed circles) concentrations. Solid lines represent fits to the data with a linear function, with slope and y intercept of 2.5 s−1μM−1 and 11.1 μM for imipramine, and 4 s−1μM−1 and 0.4 μM for astemizole, respectively. The range of drug concentrations used to fit τblock−1 data to the linear function was between 0.5 and 10 μM for imipramine and between 25 nM and 5 μM for astemizole. Symbols and associated error bars in E–G represent means ± SEM for six and seven cells for imipramine and astemizole, respectively.

Mentions: hEag1 channels do not inactivate during sustained depolarizations to potentials that activate most of the channels (Fig. 1 A, control trace). However, in the presence of imipramine (Fig. 1 A) or astemizole (Fig. 1 C), a clear time- and dose-dependent decay of hEag1 currents was observed. This suggests that both drugs block open hEag1 channels (Armstrong, 1969). After both drugs attained the equilibrium concentration near their active site, consecutive current traces recorded at 30-s intervals were identical (unpublished data). Thus, there is no trapping of imipramine and astemizole by closure of hEag1 channels (Armstrong, 1971; Choquet and Korn, 1992; Mitcheson et al., 2000a).


Mechanism of block of hEag1 K+ channels by imipramine and astemizole.

García-Ferreiro RE, Kerschensteiner D, Major F, Monje F, Stühmer W, Pardo LA - J. Gen. Physiol. (2004)

Concentration dependence of hEag1 block by imipramine and astemizole. (A and C) Superimposed hEag1 current traces recorded during 1.5 s test depolarizations to 80 mV from a holding potential of −70 mV in the absence and presence of the indicated concentrations of imipramine (Imi, A) or astemizole (Ast, C). Test potential was chosen to achieve the maximal open probability of hEag1, whose activation curve saturates above 60 mV (not depicted). The effects of drug application were monitored with test pulses applied every 30 s until a steady-state block was reached. (B and D) Current traces in the presence of imipramine or astemizole were normalized dividing them point by point by the respective preapplication traces. Solid lines indicate the best fit to a single exponential function. (E) Dose–response plots for imipramine (open circles) and astemizole (closed circles). The steady-state fraction of channels blocked was calculated from the asymptotic values of single exponential fits to current ratios as shown in B and D. Solid lines represent fits to the data using the Hill equation, with IC50 values and Hill coefficients of 1.87 μM and 1.04 for imipramine, and 0.21 μM and 1.32 for astemizole, respectively. (D) Time constant of block (τblock) for imipramine (open circles) and astemizole (closed circles) derived from the least-squares fits of single exponential functions used in E. Solid lines represent fits to the data using the Hill equations, with maximum, minimum, IC50, and Hill coefficients of 86.7 ms, 11.6 ms, 3.75 μM, and 1.27 for imipramine, and 1.33 s, 0.024 s, 0.26 μM, and 1.32 for astemizole, respectively. (G) The rate of current block is represented (τblock−1) as a linear function of nonsaturating imipramine (open circles) or astemizole (closed circles) concentrations. Solid lines represent fits to the data with a linear function, with slope and y intercept of 2.5 s−1μM−1 and 11.1 μM for imipramine, and 4 s−1μM−1 and 0.4 μM for astemizole, respectively. The range of drug concentrations used to fit τblock−1 data to the linear function was between 0.5 and 10 μM for imipramine and between 25 nM and 5 μM for astemizole. Symbols and associated error bars in E–G represent means ± SEM for six and seven cells for imipramine and astemizole, respectively.
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Related In: Results  -  Collection

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fig2: Concentration dependence of hEag1 block by imipramine and astemizole. (A and C) Superimposed hEag1 current traces recorded during 1.5 s test depolarizations to 80 mV from a holding potential of −70 mV in the absence and presence of the indicated concentrations of imipramine (Imi, A) or astemizole (Ast, C). Test potential was chosen to achieve the maximal open probability of hEag1, whose activation curve saturates above 60 mV (not depicted). The effects of drug application were monitored with test pulses applied every 30 s until a steady-state block was reached. (B and D) Current traces in the presence of imipramine or astemizole were normalized dividing them point by point by the respective preapplication traces. Solid lines indicate the best fit to a single exponential function. (E) Dose–response plots for imipramine (open circles) and astemizole (closed circles). The steady-state fraction of channels blocked was calculated from the asymptotic values of single exponential fits to current ratios as shown in B and D. Solid lines represent fits to the data using the Hill equation, with IC50 values and Hill coefficients of 1.87 μM and 1.04 for imipramine, and 0.21 μM and 1.32 for astemizole, respectively. (D) Time constant of block (τblock) for imipramine (open circles) and astemizole (closed circles) derived from the least-squares fits of single exponential functions used in E. Solid lines represent fits to the data using the Hill equations, with maximum, minimum, IC50, and Hill coefficients of 86.7 ms, 11.6 ms, 3.75 μM, and 1.27 for imipramine, and 1.33 s, 0.024 s, 0.26 μM, and 1.32 for astemizole, respectively. (G) The rate of current block is represented (τblock−1) as a linear function of nonsaturating imipramine (open circles) or astemizole (closed circles) concentrations. Solid lines represent fits to the data with a linear function, with slope and y intercept of 2.5 s−1μM−1 and 11.1 μM for imipramine, and 4 s−1μM−1 and 0.4 μM for astemizole, respectively. The range of drug concentrations used to fit τblock−1 data to the linear function was between 0.5 and 10 μM for imipramine and between 25 nM and 5 μM for astemizole. Symbols and associated error bars in E–G represent means ± SEM for six and seven cells for imipramine and astemizole, respectively.
Mentions: hEag1 channels do not inactivate during sustained depolarizations to potentials that activate most of the channels (Fig. 1 A, control trace). However, in the presence of imipramine (Fig. 1 A) or astemizole (Fig. 1 C), a clear time- and dose-dependent decay of hEag1 currents was observed. This suggests that both drugs block open hEag1 channels (Armstrong, 1969). After both drugs attained the equilibrium concentration near their active site, consecutive current traces recorded at 30-s intervals were identical (unpublished data). Thus, there is no trapping of imipramine and astemizole by closure of hEag1 channels (Armstrong, 1971; Choquet and Korn, 1992; Mitcheson et al., 2000a).

Bottom Line: Using inside- and outside-out patch recordings, we found that a permanently charged, quaternary derivative of imipramine (N-methyl-imipramine) only blocks channels from the intracellular side of the membrane.However, as astemizole competes with imipramine and intracellular TEA for binding to the channel, it is proposed to interact with an overlapping intracellular binding site.The significance of these findings, in the context of structure-function of channels of the eag family is discussed.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Molekulare Biologie Neuronaler Signale, Max-Planck Institut für Experimentelle Medizin, 37075 Göttingen, Germany. rgarcia@gwdg.de

ABSTRACT
Ether à go-go (Eag; KV10.1) voltage-gated K+ channels have been detected in cancer cell lines of diverse origin and shown to influence their rate of proliferation. The tricyclic antidepressant imipramine and the antihistamine astemizole inhibit the current through Eag1 channels and reduce the proliferation of cancer cells. Here we describe the mechanism by which both drugs block human Eag1 (hEag1) channels. Even if both drugs differ in their affinity for hEag1 channels (IC50s are approximately 2 microM for imipramine and approximately 200 nM for astemizole) and in their blocking kinetics, both drugs permeate the membrane and inhibit the hEag1 current by selectively binding to open channels. Furthermore, both drugs are weak bases and the IC50s depend on both internal an external pH, suggesting that both substances cross the membrane in their uncharged form and act from inside the cell in their charged forms. Accordingly, the block by imipramine is voltage dependent and antagonized by intracellular TEA, consistent with imipramine binding in its charged form to a site located close to the inner end of the selectivity filter. Using inside- and outside-out patch recordings, we found that a permanently charged, quaternary derivative of imipramine (N-methyl-imipramine) only blocks channels from the intracellular side of the membrane. In contrast, the block by astemizole is voltage independent. However, as astemizole competes with imipramine and intracellular TEA for binding to the channel, it is proposed to interact with an overlapping intracellular binding site. The significance of these findings, in the context of structure-function of channels of the eag family is discussed.

Show MeSH
Related in: MedlinePlus