Ethylbromide Tamoxifen, a Membrane-Impermeant Antiestrogen, Activates Smooth Muscle Calcium-Activated Large-Conductance Potassium Channels from the Extracellular Side

Abstract

Smooth-muscle calcium-activated large-conductance potassium channels (BK channels) are activated by tamoxifen and 17-␤-estradiol. This increase in NP o , the number of channels, N, multiplied by open probability, depends on the presence of the regulatory ␤1-subunit. Furthermore, a previous study indicated that 17-␤-estradiol might bind an extracellular site on the ␤1-subunit. Because tamoxifen and 17-␤-estradiol may share a common binding site, we hypothesized that tamoxifen activates BK channels through a site on the extracellular surface of the membrane. A membrane-impermeant analog of tamoxifen, ethylbromide tamoxifen, was synthesized and used to test this hypothesis in whole-cell, outside-out, cell-attached, and inside-out patches from canine colonic smooth muscle cells. Ethylbromide tamoxifen is positively charged and is therefore membrane-impermeant. In whole-cell experiments, ethylbromide tamoxifen increased K ϩ current at potentials positive to ϩ40 mV, which has previously been attributed to BK channels. Unlike tamoxifen, ethylbromide tamoxifen did not inhibit delayed rectifier current. In outside-out patches, ethylbromide tamoxifen increased BK channel NP o with an EC 50 value of 1 M. Ethylbromide tamoxifen did not increase BK channel NP o in cell-attached or inside-out patches; however, subsequent addition of equimolar tamoxifen did. Both drugs diminished BK channel unitary conductance to a degree that paralleled the effect on NP o , suggesting an additional interaction with the pore-forming ␣-subunit. An interaction of tamoxifen with the pore was supported by a right shift in the concentration-response curve for tetraethylammonium; similar results were evident with iberiotoxin and charybdotoxin block. Our data suggest that ethylbromide tamoxifen does not easily traverse the plasma membrane and that tamoxifen binding responsible for activation of BK channels is at an extracellular site. The tamoxifen binding site may be within the extracellular loop of the BK channel ␤1-subunit or, alternatively, on an as-yet-unidentified mediator that has an extracellular binding site.

Differential effects of diet-induced obesity on BKCa β1-subunit expression and function in rat skeletal muscle arterioles and small cerebral arteries

“…lished, selective activators of the BK Ca ␤ 1 -subunit (11,12,13), and in the present study tamoxifen-induced dilation was abolished by IBTX in both vessels, suggesting this vasodilation was mediated exclusively by BK Ca . In middle-cerebral arteries dilation to tamoxifen was relatively small and, combined with the relatively large constriction induced by IBTX, suggests a high level of BK Ca activity, consistent with previous studies demonstrating the importance of BK Ca in the cerebral circulation (52).…”

Use of Escin as a Perforating Agent on the IonWorks Quattro Automated Electrophysiology Platform

“…The escin pore was clearly permeable to QX314, which blocked Na V 1.7 from the intracellular side only. There are many examples in which such intracellular access to membrane-impermeant compounds might be useful (e.g., when using thiol-modifying reagents such as MTSET to study ion channel topology 21 or to study the sidedness of a compound's action, e.g., ethylbromide tamoxifen on BKCa 22 ). To conclude, this article demonstrates, for the first time, the use of escin as an alternative perforating agent on the automated electrophysiology platform IonWorks.…”

International Union of Basic and Clinical Pharmacology. XCVII. G Protein–Coupled Estrogen Receptor and Its Pharmacologic Modulators

“…Early examples include the positively charged quaternary ammonium salts of the basic amine side chain of tamoxifen that were found to exhibit similar binding affinity in cytosolic receptor extracts but greatly diminished binding and reduced cytostatic activity in the E2-responsive MCF-7 breast cancer cell line (Jarman et al, 1986). Subsequent studies have demonstrated utility and revealed potential pharmacologic applications for permanently charged tamoxifen derivatives (Biegon et al, 1996;Somjen et al, 1996;Allen et al, 2000;Dick et al, 2002;Dietze et al, 2004). The time course of experiments employing this electrostatic approach is an important consideration and should be reserved for rapid signaling events occurring with short duration (seconds-minutes), because slow passive membrane permeability is possible given longer exposure times (Rickert et al, 2010;Rivera-Guevara et al, 2010).…”