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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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pH dependence of imipramine block of hEag1 channels. (A–C) Superimposed whole-cell current traces recorded during 1-s depolarizations to 80 mV from a holding potential of −70 mV in the absence (top) and presence (bottom) of 2.5 μM imipramine. Currents were recorded in control external and internal solutions (A), or in conditions where either the pH of the external (pHext; B) or internal solutions (pHint; C) was varied to the indicated values. (D) Dose–response plots for imipramine at pHext//pHint relations of 7.4//7.35 (open circles), 6.4//7.35 (closed inverted triangles), 8.4//7.35 (closed triangles), 7.4//6.4 (open inverted triangles), and 7.4//8.4 (open upright triangles). The steady-state fraction of channels blocked was calculated as in Fig. 1 B. The data were fitted using the Hill equation (solid lines, see text for average IC50). (E) Rate of current block (τblock−1) as a function of nonsaturating imipramine concentrations recorded at pHext 7.4, and pHint 6.4 (open inverted triangles), 7.35 (closed circles), or 8.4 (open triangles). Straight lines through τblock−1 data represent fits to the data with linear functions with slopes of 12.6, 3.4, and 0.9 s−1μM−1, and y intercepts of 10.4, 10.7, and 10.7 μM to data recorded at pHint 6.4, 7.4, and 8.4, respectively. Symbols and associated error bars in D and E represent means ± SEM for three (control) and five cells (rest of the conditions). (F) logIC50 plotted as a function of the difference between pHext and pHint. Closed circles and associated error bars represent means ± SD of individual fits to cells shown in D, plus four cells tested at pHext//pHint 7.1//7.7, five cells at 7.1//6.8, three cells at 7.6//6.8, and five cells at 7.1//8. Straight line through symbols represents the best fit of a linear function with slope −0.86, and y intercept 0.43, to the data. The dotted line has the same y intercept, but a slope of −1.
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fig9: pH dependence of imipramine block of hEag1 channels. (A–C) Superimposed whole-cell current traces recorded during 1-s depolarizations to 80 mV from a holding potential of −70 mV in the absence (top) and presence (bottom) of 2.5 μM imipramine. Currents were recorded in control external and internal solutions (A), or in conditions where either the pH of the external (pHext; B) or internal solutions (pHint; C) was varied to the indicated values. (D) Dose–response plots for imipramine at pHext//pHint relations of 7.4//7.35 (open circles), 6.4//7.35 (closed inverted triangles), 8.4//7.35 (closed triangles), 7.4//6.4 (open inverted triangles), and 7.4//8.4 (open upright triangles). The steady-state fraction of channels blocked was calculated as in Fig. 1 B. The data were fitted using the Hill equation (solid lines, see text for average IC50). (E) Rate of current block (τblock−1) as a function of nonsaturating imipramine concentrations recorded at pHext 7.4, and pHint 6.4 (open inverted triangles), 7.35 (closed circles), or 8.4 (open triangles). Straight lines through τblock−1 data represent fits to the data with linear functions with slopes of 12.6, 3.4, and 0.9 s−1μM−1, and y intercepts of 10.4, 10.7, and 10.7 μM to data recorded at pHint 6.4, 7.4, and 8.4, respectively. Symbols and associated error bars in D and E represent means ± SEM for three (control) and five cells (rest of the conditions). (F) logIC50 plotted as a function of the difference between pHext and pHint. Closed circles and associated error bars represent means ± SD of individual fits to cells shown in D, plus four cells tested at pHext//pHint 7.1//7.7, five cells at 7.1//6.8, three cells at 7.6//6.8, and five cells at 7.1//8. Straight line through symbols represents the best fit of a linear function with slope −0.86, and y intercept 0.43, to the data. The dotted line has the same y intercept, but a slope of −1.

Mentions: For electrophysiological experiments, cells were grown for 24–72 h on poly-l-lysine–coated glass coverslips. All electrophysiological experiments were performed at room temperature. Macroscopic currents were recorded in the whole-cell, inside-out, or outside-out configurations of the patch-clamp technique (Hamill et al., 1981) using an EPC-9 amplifier (HEKA). Patch pipettes with a tip resistance of 0.9–1.5 MΩ were made from Corning #0010 capillary glass (WPI). Series resistance was compensated by >60%. The control internal solution contained (in mM) 100 KCl, 45 NMDG, 10 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrapotassium salt (BAPTA·K4), 10 HEPES/HCl, pH 7.35. In experiments where the pH of the internal solution was set to 6.4 or 8.4 (Figs. 8 and 9), HEPES was replaced with an equivalent concentration of MES or CHES, respectively. The control external recording solution contained (in mM) 160 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 8 glucose, 10 HEPES/NaOH, pH 7.4. In experiments where the pH of the external solution was set to 6.4 or 8.4 (Figs. 8 and 9), HEPES was replaced with an equivalent concentration of BIS-TRIS propane. In experiments using high external [K+], [Na+] was lowered so that the sum of [K+] and [Na+] remained constant. Cell-attached patches (Fig. 4) were recorded using 140 mM external K+ and a pipette solution containing control external recording solution without glucose. Inside-out patches (Figs. 9 and 10) were recorded using a pipette solution containing (in mM) 145 NaCl, 5 KCl, 3 CaCl2, 1 MgCl2, 10 HEPES/NaOH, pH 7.4, and a bath solution containing (in mM) 160 KCl, 0.5 MgCl2, 10 EGTA, 10 BIS-TRIS propane/KOH, pH 6.0–8.4.


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)

pH dependence of imipramine block of hEag1 channels. (A–C) Superimposed whole-cell current traces recorded during 1-s depolarizations to 80 mV from a holding potential of −70 mV in the absence (top) and presence (bottom) of 2.5 μM imipramine. Currents were recorded in control external and internal solutions (A), or in conditions where either the pH of the external (pHext; B) or internal solutions (pHint; C) was varied to the indicated values. (D) Dose–response plots for imipramine at pHext//pHint relations of 7.4//7.35 (open circles), 6.4//7.35 (closed inverted triangles), 8.4//7.35 (closed triangles), 7.4//6.4 (open inverted triangles), and 7.4//8.4 (open upright triangles). The steady-state fraction of channels blocked was calculated as in Fig. 1 B. The data were fitted using the Hill equation (solid lines, see text for average IC50). (E) Rate of current block (τblock−1) as a function of nonsaturating imipramine concentrations recorded at pHext 7.4, and pHint 6.4 (open inverted triangles), 7.35 (closed circles), or 8.4 (open triangles). Straight lines through τblock−1 data represent fits to the data with linear functions with slopes of 12.6, 3.4, and 0.9 s−1μM−1, and y intercepts of 10.4, 10.7, and 10.7 μM to data recorded at pHint 6.4, 7.4, and 8.4, respectively. Symbols and associated error bars in D and E represent means ± SEM for three (control) and five cells (rest of the conditions). (F) logIC50 plotted as a function of the difference between pHext and pHint. Closed circles and associated error bars represent means ± SD of individual fits to cells shown in D, plus four cells tested at pHext//pHint 7.1//7.7, five cells at 7.1//6.8, three cells at 7.6//6.8, and five cells at 7.1//8. Straight line through symbols represents the best fit of a linear function with slope −0.86, and y intercept 0.43, to the data. The dotted line has the same y intercept, but a slope of −1.
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Related In: Results  -  Collection

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fig9: pH dependence of imipramine block of hEag1 channels. (A–C) Superimposed whole-cell current traces recorded during 1-s depolarizations to 80 mV from a holding potential of −70 mV in the absence (top) and presence (bottom) of 2.5 μM imipramine. Currents were recorded in control external and internal solutions (A), or in conditions where either the pH of the external (pHext; B) or internal solutions (pHint; C) was varied to the indicated values. (D) Dose–response plots for imipramine at pHext//pHint relations of 7.4//7.35 (open circles), 6.4//7.35 (closed inverted triangles), 8.4//7.35 (closed triangles), 7.4//6.4 (open inverted triangles), and 7.4//8.4 (open upright triangles). The steady-state fraction of channels blocked was calculated as in Fig. 1 B. The data were fitted using the Hill equation (solid lines, see text for average IC50). (E) Rate of current block (τblock−1) as a function of nonsaturating imipramine concentrations recorded at pHext 7.4, and pHint 6.4 (open inverted triangles), 7.35 (closed circles), or 8.4 (open triangles). Straight lines through τblock−1 data represent fits to the data with linear functions with slopes of 12.6, 3.4, and 0.9 s−1μM−1, and y intercepts of 10.4, 10.7, and 10.7 μM to data recorded at pHint 6.4, 7.4, and 8.4, respectively. Symbols and associated error bars in D and E represent means ± SEM for three (control) and five cells (rest of the conditions). (F) logIC50 plotted as a function of the difference between pHext and pHint. Closed circles and associated error bars represent means ± SD of individual fits to cells shown in D, plus four cells tested at pHext//pHint 7.1//7.7, five cells at 7.1//6.8, three cells at 7.6//6.8, and five cells at 7.1//8. Straight line through symbols represents the best fit of a linear function with slope −0.86, and y intercept 0.43, to the data. The dotted line has the same y intercept, but a slope of −1.
Mentions: For electrophysiological experiments, cells were grown for 24–72 h on poly-l-lysine–coated glass coverslips. All electrophysiological experiments were performed at room temperature. Macroscopic currents were recorded in the whole-cell, inside-out, or outside-out configurations of the patch-clamp technique (Hamill et al., 1981) using an EPC-9 amplifier (HEKA). Patch pipettes with a tip resistance of 0.9–1.5 MΩ were made from Corning #0010 capillary glass (WPI). Series resistance was compensated by >60%. The control internal solution contained (in mM) 100 KCl, 45 NMDG, 10 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrapotassium salt (BAPTA·K4), 10 HEPES/HCl, pH 7.35. In experiments where the pH of the internal solution was set to 6.4 or 8.4 (Figs. 8 and 9), HEPES was replaced with an equivalent concentration of MES or CHES, respectively. The control external recording solution contained (in mM) 160 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 8 glucose, 10 HEPES/NaOH, pH 7.4. In experiments where the pH of the external solution was set to 6.4 or 8.4 (Figs. 8 and 9), HEPES was replaced with an equivalent concentration of BIS-TRIS propane. In experiments using high external [K+], [Na+] was lowered so that the sum of [K+] and [Na+] remained constant. Cell-attached patches (Fig. 4) were recorded using 140 mM external K+ and a pipette solution containing control external recording solution without glucose. Inside-out patches (Figs. 9 and 10) were recorded using a pipette solution containing (in mM) 145 NaCl, 5 KCl, 3 CaCl2, 1 MgCl2, 10 HEPES/NaOH, pH 7.4, and a bath solution containing (in mM) 160 KCl, 0.5 MgCl2, 10 EGTA, 10 BIS-TRIS propane/KOH, pH 6.0–8.4.

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