European Journal of Pharmacology 605 (2009) 36–45

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European Journal of Pharmacology

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Molecular and Cellular Pharmacology Structural determinants of lolitrems for inhibition of BK large conductance Ca2+-activated K+ channels☆

Wendy L. Imlach a,c,1, Sarah C. Finch b, James Dunlop a, Julie E. Dalziel a,⁎ a AgResearch, Grasslands Research Centre, Palmerston North 4442, New Zealand b AgResearch, Ruakura Research Centre, Hamilton 3240, New Zealand c Department of Pharmacology and , University of Otago, Dunedin, New Zealand article info abstract

Article history: is an -diterpenoid which is the main causative agent of ryegrass staggers, an Received 20 October 2008 animal disease associated with tremors and incoordination. It is also a potent inhibitor of large conductance Received in revised form 12 December 2008 calcium-activated potassium (BK) channel activity (IC50 =4 nM). Furthermore, we have recently shown that Accepted 23 December 2008 the motor function deficits induced by lolitrem B are specifically mediated by BK channels, making the Available online 10 January 2009 a valuable tool for investigating the molecular function and physiological roles of these channels. To determine what structural features of BK channel agents are required for high potency, the effect of lolitrem B Keywords: – Large conductance calcium-activated and seven structurally-related lolitrems on BK channel activity has been measured. Concentration responses and conductance–voltage (G–V) relationships were determined for each compound and related to the BK channel different structure types. This study has identified seven new BK channel inhibitors and has allowed the Patch-clamp identification of two key structural features required for high potency BK channel activity by lolitrems. Fungal toxin © 2009 Elsevier B.V. All rights reserved. Indole diterpene Lolitrem

1. Introduction gical roles vary greatly among tissues, the molecular function of BK channels at a cellular level is generally to limit cellular activity. When Large conductance calcium-activated potassium (BK) ion channels activated, BK channels permit the flow of K+ out of the cell which has a are expressed in cell membranes of virtually every tissue in the body. hyperpolarising influence on the resting membrane potential. Thus they They have been attributed roles in a growing number of physiological tend to regulate physiological processes by preventing over-excitation, processes as new genetic and pharmacological tools have led to although this varies among tissues as the effect on cellular excitability improved understanding of their molecular function and regulation. depends on the calcium-dependence of the BK channel type and is BK channels have major roles in smooth muscle function that include: influenced by the other ion channels present. Malfunction of BK regulation of blood pressure, urinary bladder function (Brenner et al., channels contributes to disease processes such as: hypertension 2000b; Meredith et al., 2004; Thorneloe et al., 2005), gut (Sausbier et al., (Amberg and Santana, 2003; Brenner et al., 2000b), epilepsy (Du et al., 2006) and erectile function (Werner et al., 2005). In the brain, BK 2005), and diabetic retinopathy (McGahon et al., 2007). Given their roles channels have roles in motor function (Imlach et al., 2008; Meredith in many pathophysiological conditions, BK channels are considered a et al., 2004; Sausbier et al., 2004), circadian rhythm (Meredith et al., promising target for new drugs (Nardi and Olesen, 2008). 2006), cerebrovascular circulation (Filosa et al., 2006), and neuronal BK channel blockers have been valuable pharmacological tools for circuits (Brenner et al., 2005). Other physiological roles of BK channels unravelling structure, mechanism and the physiological roles of BK include: hearing (Pyott et al., 2007; Ruttiger et al., 2004) immune channels (Ghatta et al., 2006). These include the peptide pore blockers function (Ahluwalia et al., 2004), and protection from the effects of iberiotoxin and from , quaternary ischemia in cardiac mitochondria (Xu et al., 2002). While its physiolo- ammonium compounds, for example (TEA), and non-peptide fungal alkaloids. Since the peptide inhibitors are

☆ This work was supported by the Marsden Fund, Royal Society of New Zealand, grant membrane impermeable they have limited use in whole animal AGR302 (J.E.D. and S.F). studies making the non-peptide blockers particularly valuable. The ⁎ Corresponding author. AgResearch, Grasslands Research Centre, Tennent Drive, indole-diterpenoid, , isolated from the fungus Penicillium Private Bag 11008, Palmerston North 4442, New Zealand. Tel.: +64 6 3518098; fax: +64 6 paxilli has proved useful for both in vitro and in vivo studies, but in 3518032. view of the importance of BK channels, new inhibitors compatible E-mail address: [email protected] (J.E. Dalziel). 1 Present address: Columbia University, Medical Center, Department of Physiology with in vivo studies would be very valuable. These compounds would and Cellular Biophysics, New York, NY 10032, USA. give new molecular tools to impair BK channel function and thus

0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.12.031 W.L. Imlach et al. / European Journal of Pharmacology 605 (2009) 36–45 37 elucidate their roles in different tissues. We have recently discovered through inhibition of BK channels (Imlach et al., 2008). Lolitrem B is that lolitrem B, another fungal alkaloid, has an apparent affinity for BK therefore not only a potent BK channel inhibitor but it is also specific channels 5 times higher than that of paxilline (Imlach et al., 2008). for this channel type, in that high doses have no effect on motor Originating in planta, lolitrem B is a product of the grass– function in BK channel knockout mice (Imlach et al., 2008), and is able symbiotic relationship (Lane et al., 2000). Perennial ryegrass infected to be used in whole animal studies. This compound therefore provides with the endophytic fungus Neotyphodium lolii produces secondary an additional pharmacological tool to investigate BK channel function. metabolites that include the lolitrem family of indole diterpenes. Of At present, it is not known where lolitrem B binds to BK channels, or these, the predominant compound, lolitrem B, is considered the main which structural features of the inhibitor confer its high apparent toxin responsible for a neurological condition called ryegrass staggers affinity for BK channels. which causes tremors and impairs motor function in grazing animals The aim of this study was to determine which components of the (Gallagher and Hawkes, 1986). Using a mouse model, we have recently lolitrem structure are important for BK channel inhibition. To achieve shown that motor function deficits induced by lolitrem B are mediated this, we isolated four lolitrem compounds from endophyte-infected

Fig. 1. Comparison of BK channel inhibition by A/B ring junction stereoisomers of lolitrem B. (A) Chemical structures of lolitrem B, lolitrem F and 31-epilolitrem B. (B) Representative K+ currents recorded from hSlo inside-out macropatches from HEK cells are shown at a range of concentrations for each corresponding compound. A 20 ms depolarising pulse to +150 mV was applied from a holding potential of −80 mV in 10 μM free calcium. The zero current level is indicated by a broken line. (C) Concentration–response relationship for hSlo current inhibition by lolitrem B (circles, n=19), lolitrem F (squares, n=10), and 31-epilolitrem B (triangles, n=10) shown as a fraction of the control response. IC50 values are given in Table 1. Download English Version: https://daneshyari.com/en/article/2534656

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