Molecular and cellular pharmacologyCO-independent modification of K+ channels by tricarbonyldichlororuthenium(II) dimer (CORM-2)
Graphical abstract
Introduction
Carbon monoxide (CO) is an odorless toxic gas, typically generated during incomplete combustion of organic matter. However, CO is also endogenously produced in mammals (Tenhunen et al., 1968) and affects many processes such as vascular tone regulation (Coburn, 1979) and synaptic transmission (Zhuo et al., 1993). Therefore, CO may also serve as a physiological gaseous messenger with therapeutic potential. This aspect spurred the development of CO-releasing molecules (CORMs) to be used as drugs, circumventing complications during therapeutic inhalation of CO gas.
Among several different CORMs synthesized, tricarbonyldichlororuthenium(II) dimer (CORM-2; Motterlini et al., 2002), most likely because of its commercial availability, has been used extensively in in-vitro studies. Compared with application of CO itself, CORMs are safer and easier to use in experimental settings; however, a drawback of using CORMs is the potential problem of eliciting molecular reactions that are unrelated to CO itself but originate from other by-products. Unfortunately, such CORM-mediated side effects have not been studied systematically.
Studies utilizing CORMs have implicated numerous molecular effectors of CO (reviewed in e.g. Gullotta et al., 2012; Wegiel et al., 2013). For example, it is generally accepted that activation of large-conductance, Ca2+- and voltage-activated K+ (KCa1.1) channels contributes to the CO-dependent vasorelaxation (Wang et al., 1997, Williams et al., 2004). However, vasorelaxation induced by CO gas and CORM-2 apparently involves different molecular mechanisms (Decaluwé et al., 2012). Furthermore, CO-mediated activation of KCa1.1 channels in human umbilical vein endothelial cells is not mimicked by CORM-2 (Dong et al., 2008).
The tetrameric KCa1.1 channels are composed of a transmembrane central pore domain surrounded by four voltage-sensing domains, similar to voltage-gated K+ (Kv) channels. Two large cytosolic C-terminal domains (RCK1 and RCK2), which are absent in Kv channels, form a gating ring structure. The channel open probability is controlled by transmembrane voltage and the conformation of the gating ring, which changes upon binding of intracellular Ca2+ (Hoshi et al., 2013) or a plethora of molecules, among them possibly CO (Hou et al., 2009). Activating impacts of CO gas or several CORMs have been reported, but the underlying molecular mechanisms are still under debate. Proposed molecular determinants for CO effects on KCa1.1 include extracellular histidines (Wang and Wu, 1997), channel-bound heme (Jaggar et al., 2005), H365 and H394 within RCK1 (Hou et al., 2008b), and C911 within RCK2 (Williams et al., 2008, Telezhkin et al., 2011).
Here we analyzed the mechanism by which CORM-2 – as compared to CO gas – affects KCa1.1, Kv11.1 (hERG1) and Kv1.5 channels. We present generally applicable experimental strategies for identifying and avoiding side effects originating from CORM-2 and related CO-releasing compounds.
Section snippets
Expression plasmids and mutagenesis
Wild-type human K+ channels used in this study were: KCa1.1, (hSlo1, KCNMA1, U11058), Kv1.5 (KCNA5, P22460), Kv10.1 (hEAG1, KCNH1, AJ0013668), Kv11.1 (hERG1, KCNH2, NM_000238), and Kv11.3 (hERG3, KCNH7, NP_150375). Mutations were introduced by overlap extension PCR (Expand High Fidelity, Roche, Mannheim, Germany), verified by DNA sequencing.
Cell culture
HEK 293T cells (DSMZ, Braunschweig, Germany) were maintained in DMEM/F-12 (Life Technologies, Darmstadt, Germany) supplemented with 10% fetal bovine serum
CORM-2 activates KCa1.1 channels independently of CO
Quantitative evaluation of how CORM-2 as compared to CO dissolved in the intracellular solution activates KCa1.1 channels was performed after transient expression of KCNMA1 (hSlo1) in HEK 293T cells. K+ current was recorded in the inside-out patch-clamp configuration in the virtual absence of intracellular Ca2+ during repetitive depolarizing voltage steps; the impact of 50 µM CORM-2, which should be capable of releasing up to 300 µM CO, and that of intracellular buffer with an estimated
Discussion
Physiologically produced CO, mainly by the catabolism of heme catalyzed by heme oxygenase, is a putative cellular messenger. However, unlike NO, which has been studied extensively and has clearly identified molecular targets, the medicinal benefits of CO and the underlying molecular mechanisms are less well defined. For possible clinical applications and for studying CO-related biology, the local release of CO by means of CORMs is desirable. However, potential confounding factors must be
Acknowledgments
This work was supported by the German Research Foundation (DFG, FOR 1738 and HE2993/16-1) and the National Institutes of Health (to TH, GM121375). We furthermore thank M. Keating for providing cDNA coding for KCNH2 and E.M. Franke for technical assistance.
Statement of conflicts of interest
The authors declare no competing interests.
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