Molecular and Cellular Pharmacology Phenformin has a direct inhibitory effect on the ATP-sensitive potassium channel Qadeer Aziz, Alison Thomas, Tapsi Khambra, Andrew Tinker ⁎ Department of Medicine, University College London, Rayne Institute, 5 University Street, London, WC1E 6JJ, United Kingdom abstract article info Article history: Received 19 October 2009 Received in revised form 29 January 2010 Accepted 14 February 2010 Available online 25 February 2010 Keywords: K ATP channel Phenformin AMPK Vascular smooth muscle cell Whole-cell patch-clamp The biguanides, phenformin and metformin, are used in the treatment of type II diabetes mellitus, as well as being routinely used in studies investigating AMPK activity. We used the patch-clamp technique and rubidium flux assays to determine the role of these drugs in ATP-sensitive K + channel (K ATP ) regulation in cell lines expressing the cloned components of K ATP and the current natively expressed in vascular smooth muscle cells (VSMCs). Phenformin but not metformin inhibits a number of variants of K ATP including the cloned equivalents of currents present in vascular and non-vascular smooth muscle (Kir6.1/SUR2B and Kir6.2/SUR2B) and pancreatic β-cells (Kir6.2/SUR1). However it does not inhibit the current potentially present in cardiac myocytes (Kir6.2/SUR2A). The highest affinity interaction is seen with Kir6.1/SUR2B (IC 50 = 0.55 mM) and it also inhibits the current in native vascular smooth muscle cells. The extent and rate of inhibition are similar to that seen with the known K ATP blocker PNU 37883A. Additionally, phenformin inhibited the current elicited through the Kir6.2ΔC26 (functional without SUR) channel with an IC 50 of 1.78 mM. Phenformin reduced the open probability of Kir6.1/SUR2B channels by ∼ 90% in inside-out patches. These findings suggest that phenformin interacts directly with the pore-forming Kir6.0 subunit however the sulphonylurea receptor is able to significantly modulate the affinity. It is likely to block from the intracellular side of the channel in a manner analogous to that of PNU 37883A. © 2010 Elsevier B.V. All rights reserved. 1. Introduction K ATP channels are present in many cell types and couple cell metabolism to potassium flux across cell membranes (Rodrigo and Standen, 2005). Metabolic regulation of K ATP is mediated via cellular changes in ATP, where increased or decreased ATP concentrations result in inhibition and activation of K ATP , respectively. In addition, rising ADP as a result of a metabolic challenge such as ischaemia and hypoxia also activate the channel (Rodrigo and Standen, 2005). K ATP channels are composed of an octameric complex of pore- forming subunits (Kir6.1 or Kir6.2), members of the inwardly rectifying potassium channel family, and the sulphonylurea receptor subunit (SUR1, SUR2A and SUR2B) who are members of the ATP- binding cassette family of proteins (Seino, 1999; Rodrigo and Standen, 2005). Co-assembly of a particular Kir6 with a particular SUR in different tissues generates currents that have a characteristic conductance, nucleotide regulation and pharmacology (Seino, 1999; Babenko et al., 1998; Tucker and Ashcroft, 1998). For example, Kir6.1 with SUR2B is thought to comprise the vascular smooth muscle K ATP current (Yamada et al., 1997; Beech et al., 1993; Cui et al., 2002; Miki et al., 2002), Kir6.2 with SUR2B is present in non-vascular smooth muscle (Isomoto et al., 1996), Kir6.2 with SUR2A or SUR1 comprise the cardiac K ATP channel (Alekseev et al., 2005) and the pancreatic K ATP channel respectively (Aguilar-Bryan et al., 1995; Inagaki et al., 1995). Oral biguanides and metformin in particular are used in the treatment of type II diabetes mellitus. Phenformin was withdrawn from clinical use in the 1970s as it was associated with fatal lactic acidosis. They act by sensitising peripheral tissues to insulin and inhibiting hepatic gluconeogenesis. Furthermore, metformin and phenformin are used as activators of AMP-activated protein kinase (AMPK) and a number of their cellular effects are probably mediated through this action (Zhou et al., 2001). Indeed, these drugs are routinely used to investigate AMPK involvement in cell signalling (Hardie, 2008). AMPK, a serine/threonine kinase, coordinates cellular energy metabolism and ATP synthesis/conservation with metabolic demand to regulate the energy balance within the cell (Hardie and Carling, 1997). It is activated by an increased AMP:ATP ratio and subsequently acts on various downstream targets (Hardie, 2004). It has been reported that AMPK can couple membrane transport with cellular metabolism by inhibiting some ion transporters such as cystic fibrosis transmembrane regulator (CFTR) and the epithelial sodium channel in order to promote cell survival (Hallows et al., 2003; Woollhead et al., 2005). More recently, the presence of AMPK has been shown to promote surface expression of cardiac K ATP channels and to be important during preconditioning of cardiomyocytes (Sukhodub et al., 2007). European Journal of Pharmacology 634 (2010) 26–32 ⁎ Corresponding author. Tel.: + 44 20 7679 6391. E-mail address: a.tinker@ucl.ac.uk (A. Tinker). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.02.023 Contents lists available at ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar