Mechanism of BK Channel Inhibition by the Opioid Agonist Loperamide

Abstract

Large conductance Ca2+-activated K+ (BK) channels are expressed in a variety of tissues and control a range of physiological functions, including action potential duration in some neurons and contractility in smooth muscle. Pharmacological BK channel modulators may be promising therapeutic options in human disease; however, the current library of effective BK-selective modulators is limited. Using a high-throughput screening approach, we observed that the diphenylpropylamine μ-opioid agonist, loperamide, could inhibit BK channel activity. Here we analyze the molecular mechanism of this inhibition using patch-clamp electrophysiology. In excised inside-out patch recordings (Ca2+ = 88 μM at the cytosolic side), loperamide reversibly inhibited BK current in a dose-dependent manner when applied to the cytosolic face of BK channels, with an IC50 of 1.0 ± 0.2 μM. Over a range of voltages at which BK channels were open nearly 100% of the time, the IC50 did not significantly increase or decrease as a function of voltage, suggesting that loperamide does not inhibit as an open-channel blocker. Single-channel recordings revealed that loperamide does not reduce unitary current amplitudes, and does not inhibit BK channels at voltages where voltage sensors were at rest (between −140 and −160 mV; Po = 0.0035 ± 0.0002 and Po = 0.0033 ± 0.0001 with 0 and 30 μM loperamide, respectively). Loperamide inhibited BK channels in the nominal absence of Ca2+, in channels co-expressed with the γ1 subunit (LRRC26), suggesting that inhibition does not strictly depend on the presence of Ca2+. Together, these data are consistent with the idea that loperamide inhibits BK channels either through directly inhibiting activation of the BK voltage sensor, or by inhibiting allosteric coupling between voltage sensor activation and pore opening. This work has been supported by NIH grant R01 GM126581 and AHA Grant-in-Aid to B.S.R.