Elsevier

Toxicology in Vitro

Volume 21, Issue 4, June 2007, Pages 656-664
Toxicology in Vitro

Inhibition by cocaine of G protein-activated inwardly rectifying K+ channels expressed in Xenopus oocytes

https://doi.org/10.1016/j.tiv.2007.01.009Get rights and content

Abstract

Cocaine, a commonly abused psychostimulant, interacts with not only transporters for dopamine, serotonin and norepinephrine but also several receptors and channels. However, the molecular mechanisms underlying the various effects of cocaine remain to be clarified. Using the Xenopus oocyte expression assay, we investigated the effects of cocaine on G protein-activated inwardly rectifying K+ (GIRK) channels, which regulate neuronal excitability and the heart rate. In oocytes injected with mRNAs for GIRK1/GIRK2, GIRK2 or GIRK1/GIRK4 subunits, cocaine reversibly reduced basal GIRK inward currents. The inhibition by cocaine at the toxic levels was concentration-dependent, but voltage-independent and time-independent during each voltage pulse. However, methylphenidate, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) at their toxic concentrations had little effect on the channels. Additionally, Kir1.1 and Kir2.1 channels were insensitive to all of the drugs. The inhibition by cocaine, which exists mainly in a protonated form at pH 7.4, was not affected by extracellular pH 9, at which the proportion of the uncharged form increases, suggesting the inhibition by both forms with similar effectiveness, and at physiological pH the effect being predominantly due to the protonated cocaine. Our results suggest that inhibition of GIRK channels by cocaine may contribute to some of its toxic effects.

Introduction

Cocaine is well known as an abused psychostimulant drug (O’Brien, 2001). Also, cocaine clinically exerts its local anesthetic effect by blocking voltage-gated Na+ channels, but its clinical use has decreased because of toxicity, namely, cardiovascular, neurologic and psychiatric complications such as arrhythmias, seizures and delirium, and its properties leading to addiction and abuse (O’Brien, 2001, White and Lambe, 2003). Cocaine binds transporters for dopamine, serotonin and norepinephrine (DAT, SERT and NET, respectively), and inhibits the reuptake of monoamines (O’Brien, 2001). The resulting potentiation of the monoaminergic neurotransmission systems is thought to play an important role in the physiological and behavioral effects (O’Brien, 2001). It has also been shown that cocaine interacts with several receptors and channels, namely, nicotinic acetylcholine (Karpen et al., 1982), γ-aminobutyric acid type A (GABAA), glycine (Ye et al., 1999, Ren et al., 1999), serotonin type 3 (5-HT3) (Breitinger et al., 2001), muscarinic (Sharkey et al., 1988b) and σ (Sharkey et al., 1988a) receptors, and voltage-gated Ca2+ and K+ channels (Kimura et al., 1992; Premkumar, 2005). These actions of cocaine with relatively low potencies might also be involved in the molecular mechanisms underlying some of the various pharmacological effects of cocaine.

G protein-activated inwardly rectifying K+ (GIRK) channels (also known as Kir3 channels) are members of a family of inwardly rectifying K+ (Kir) channels that includes seven subfamilies (Reimann and Ashcroft, 1999). Four GIRK channel subunits have been identified in mammals (Kubo et al., 1993b; Lesage et al., 1995, Krapivinsky et al., 1995). Neuronal GIRK channels are predominantly heteromultimers composed of GIRK1 and GIRK2 subunits in most brain regions or homomultimers composed of GIRK2 subunits in the substantia nigra and ventral tegmental area (Kobayashi et al., 1995, Lesage et al., 1995, Liao et al., 1996), whereas atrial GIRK channels are heteromultimers composed of GIRK1 and GIRK4 subunits (Krapivinsky et al., 1995). A variety of G-protein-coupled receptors, such as M2 muscarinic, α2 adrenergic, D2 dopaminergic, 5-HT1A, μ-, δ- and κ-opioid, nociceptin/orphanin FQ and A1 adenosine receptors, activate GIRK channels through the direct action of G protein βγ-subunits released from Gi/o proteins (Dascal, 1997, Kobayashi and Ikeda, 2006). In addition, ethanol activates GIRK channels independently of G-protein-coupled signaling pathways (Kobayashi et al., 1999). Activation of GIRK channels causes membrane hyperpolarization, and thus the channels play an important role in reducing neuronal excitability and the heart rate (Dascal, 1997, Signorini et al., 1997, Bettahi et al., 2002). Previously, it was shown that bupivacaine and lidocaine, local anesthetics, inhibited cloned GIRK channels expressed in Xenopus oocytes (Zhou et al., 2001). Cocaine, which is structurally related to these local anesthetics, inhibited muscarinic K+ currents in ferret cardiac myocytes (Xiao and Morgan, 1998), suggesting inhibition of GIRK channels in the heart. In the present study, we investigated the effects of cocaine on brain-type and cardiac-type GIRK channels composed of cloned GIRK subunits by using the Xenopus oocyte expression assay. Furthermore, the effects of some of chemically different psychostimulants: methamphetamine (MAP), 3,4-methylenedioxymethamphetamine (MDMA) and methylphenidate (MPH), on GIRK channels were also examined.

Section snippets

Preparation of specific mRNAs

Plasmids containing the entire coding sequences for the mouse GIRK1, GIRK2, and GIRK4 channel subunits were obtained by using the polymerase chain reaction method as described previously (Kobayashi et al., 1995, Kobayashi et al., 1999). In addition, cDNAs for rat Kir1.1 in pSPORT and mouse Kir2.1 in pcDNA1 were provided by Dr. Steven C. Hebert and Dr. Lily Y. Jan, respectively (Ho et al., 1993, Kubo et al., 1993a). These plasmids were linearized by digestion with the appropriate enzyme as

Inhibition of GIRK channels by psychostimulants

In Xenopus oocytes co-injected with GIRK1 and GIRK2 mRNAs, basal GIRK currents (Kobayashi et al., 2004), which are known to depend on free G protein βγ-subunits present in the oocytes because of the inherent activity of G proteins (Dascal, 1997), were observed under the conditions of a hK solution containing 96 mM K+ and negative membrane potentials (1547.7 ± 175.6 nA at −70 mV, n = 7, Fig. 1a). Application of 500 μM cocaine immediately and reversibly caused a reduction in the inward currents through

Discussion

The present study demonstrated that cocaine inhibited brain-type GIRK1/2 and GIRK2 channels and cardiac-type GIRK1/4 channels at micromolar concentrations or more. The inhibition of GIRK1/2 heteromeric channels, which are the predominant GIRK channels present in the brain, was more potent than that of GIRK2 and GIRK1/4 channels. Autopsy studies have shown that the serum concentrations of cocaine were approximately 20 μM on the average with a wide range (Mittleman and Wetli, 1984), and reached up

Acknowledgments

We thank Dr. Steven C. Hebert (Yale University) and Dr. Lily Y. Jan (University of California, San Francisco) for generously providing the Kir1.1 cDNA and Kir2.1 cDNA, respectively. We also thank Dr. Kansaku Baba for his cooperation. This work was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Ministry of Health, Labour and Welfare of Japan.

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