Tertiapin-Q Blocks Recombinant and Native Large Conductance K + Channels in a Use-Dependent Manner Refik Kanjhan, Elizabeth J. Coulson, David J. Adams, and Mark C. Bellingham School of Biomedical Sciences (R.K., E.J.C., D.J.A., M.C.B.) and The Queensland Brain Institute (E.J.C.), University of Queensland, Brisbane, Australia Received March 7, 2005; accepted June 2, 2005 ABSTRACT Tertiapin, a short peptide from honey bee venom, has been reported to specifically block the inwardly rectifying K + (Kir) channels, including G protein-coupled inwardly rectifying po- tassium channel (GIRK) 1+GIRK4 heteromultimers and ROMK1 homomultimers. In the present study, the effects of a stable and functionally similar derivative of tertiapin, tertiapin-Q, were ex- amined on recombinant human voltage-dependent Ca 2+ -acti- vated large conductance K + channel (BK or MaxiK; -subunit or hSlo1 homomultimers) and mouse inwardly rectifying GIRK1+GIRK2 (i.e., Kir3.1 and Kir3.2) heteromultimeric K + channels expressed in Xenopus oocytes and in cultured newborn mouse dorsal root ganglion (DRG) neurons. In two- electrode voltage-clamped oocytes, tertiapin-Q (1–100 nM) in- hibited BK-type K + channels in a use- and concentration- dependent manner. We also confirmed the inhibition of recombinant GIRK1+GIRK2 heteromultimers by tertiapin-Q, which had no effect on endogenous depolarization- and hyper- polarization-activated currents sensitive to extracellular diva- lent cations (Ca 2+ , Mg 2+ , Zn 2+ , and Ba 2+ ) in defolliculated oocytes. In voltage-clamped DRG neurons, tertiapin-Q voltage- and use-dependently inhibited outwardly rectifying K + cur- rents, but Cs + -blocked hyperpolarization-activated inward cur- rents including I H were insensitive to tertiapin-Q, baclofen, bar- ium, and zinc, suggesting absence of functional GIRK channels in the newborn. Under current-clamp conditions, tertiapin-Q blocked the action potential after hyperpolarization (AHP) and increased action potential duration in DRG neurons. Taken together, these results demonstrate that the blocking actions of tertiapin-Q are not specific to Kir channels and that the block- ade of recombinant BK channels and native neuronal AHP currents is use-dependent. Inhibition of specific types of Kir and voltage-dependent Ca 2+ -activated K + channels by tertiapin-Q at nanomolar range via different mechanisms may have implications in pain physiology and therapy. K + channels form the largest family of mammalian mem- brane ion channels and play a pivotal role in function or dysfunction of excitable cells (Hille, 2001). Thus, there is substantial interest in identifying specific modulators of these ion channels. Tertiapin, a 21-amino acid-containing peptide isolated from the venom of the European honey bee (Gauldie et al., 1976; Xu and Nelson, 1993), has been char- acterized as a specific and potent blocker of recombinant GIRK1+GIRK4 heteromultimers and ROMK1 at nanomolar affinity (Jin and Lu, 1998) and ROMK2 at micromolar range (Sackin et al., 2003). On the other hand, tertiapin has been reported to be less effective on some Kir channels; for exam- ple, inhibition of Kir2.1 channels by 1 M tertiapin is less than 10% (Jin and Lu, 1998). Tertiapin-Q is a stable (non- oxidizable) and functionally similar derivative of tertiapin whose methionine residue 13 is replaced by a glutamine (Jin and Lu, 1999). The mechanism of tertiapin-Q inhibition of Kir channels is not well understood, but it is believed to block the K + channel pore by occluding its  helix into the channel vestibule (Jin et al., 1999). Kirs are highly K + -selective ion channels that carry K + from the extracellular to the intra- cellular compartment more efficiently than the opposite di- rection (Yamada et al., 1998). This inwardly rectifying prop- erty allows the channel to set the resting membrane potential toward the K + equilibrium potential without pre- venting action potential generation (Yamada et al., 1998; Hille, 2001). The action potential in mammalian ganglion neurons is terminated by fast inactivation of an inward Na + current and also by activation of outward K + currents, including tran- sient outward (I A ), delayed rectifier (I Kv ), and Ca 2+ -depen- This work was supported by the Australian Research Council (DP0208295), by the Australian National Health and Medical Research Council (210256), and by the Motor Neuron Disease Research Institute of Australia. D.J.A. and M.C.B. contributed equally to this work. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.085928. ABBREVIATIONS: GIRK, G protein-coupled inwardly rectifying potassium channel; Kir, inwardly rectifying K + channel; AHP, afterhyperpolariza- tion; SK, small conductance K + channel; IK, intermediate conductance K + channel; BK, large conductance K + channel; DRG, dorsal root ganglion; P0, postnatal day 0. 0022-3565/05/3143-1353–1361$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 314, No. 3 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 85928/3047244 JPET 314:1353–1361, 2005 Printed in U.S.A. 1353 at ASPET Journals on February 5, 2016 jpet.aspetjournals.org Downloaded from