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Inhibition of G protein-activated inwardly rectifying K+ channels by fluoxetine (Prozac) - PubMed

Inhibition of G protein-activated inwardly rectifying K+ channels by fluoxetine (Prozac)

Toru Kobayashi et al. Br J Pharmacol. 2003 Mar.

Abstract

1. The effects of fluoxetine, a commonly used antidepressant drug, on G protein-activated inwardly rectifying K(+) channels (GIRK, Kir3) were investigated using Xenopus oocyte expression assays. 2. In oocytes injected with mRNAs for GIRK1/GIRK2, GIRK2 or GIRK1/GIRK4 subunits, fluoxetine reversibly reduced inward currents through the basal GIRK activity. The inhibition by fluoxetine showed a concentration-dependence, a weak voltage-dependence and a slight time-dependence with a predominant effect on the instantaneous current elicited by voltage pulses and followed by slight further inhibition. Furthermore, in oocytes expressing GIRK1/2 channels and the cloned Xenopus A(1) adenosine receptor, GIRK current responses activated by the receptor were inhibited by fluoxetine. In contrast, ROMK1 and IRK1 channels in other Kir channel subfamilies were insensitive to fluoxetine. 3. The inhibitory effect on GIRK channels was not obtained by intracellularly applied fluoxetine, and not affected by extracellular pH, which changed the proportion of the uncharged to protonated fluoxetine, suggesting that fluoxetine inhibits GIRK channels from the extracellular side. 4. The GIRK currents induced by ethanol were also attenuated in the presence of fluoxetine. 5. We demonstrate that fluoxetine, at low micromolar concentrations, inhibits GIRK channels that play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate through activation of various G-protein-coupled receptors. The present results suggest that inhibition of GIRK channels by fluoxetine may contribute to some of its therapeutic effects and adverse side effects, particularly seizures in overdose, observed in clinical practice.

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Figures

**Figure 1**
Inhibition by fluoxetine of brain-type GIRK1/2 channels and cardiac-type GIRK1/4 channels expressed in *Xenopus* oocytes. (a) *Top*: In an oocyte coinjected with GIRK1 and GIRK2 mRNAs, current responses to 10 μ
M
fluoxetine and 10 μ
M
fluoxetine in the presence of 3 m
M
Ba²⁺. *Middle*: In an oocyte coinjected with GIRK1 and GIRK4 mRNAs, current responses to 10 μ
M
fluoxetine and 3 mM Ba²⁺. *Bottom*: In an uninjected oocyte, no significant current responses to 200 μ
M
fluoxetine and 3 m
M
Ba²⁺. Asterisks show the zero current level. Bars show the duration of application. (b) Concentration–response relations for fluoxetine in regard to the effects on GIRK1/2 channels (left) and GIRK1/4 channels (right). The magnitudes of inhibition of GIRK current by fluoxetine were compared with the 3 m
M
Ba²⁺-sensitive current components, which were 741.6±118.0 nA (n=5) in oocytes expressing GIRK1/2 channels and 600.8±268.0 nA (n=5) in oocytes expressing GIRK1/4 channels, respectively. Current responses were measured at a membrane potential of −70 mV in a high-potassium solution containing 96 m
M
K⁺ (pH 7.4). Each point and error bar represents the mean and s.e.m. of the percentage responses obtained from five oocytes. Data points were fitted using a logistic equation.

**Figure 2**
Characteristics in the effects of fluoxetine on GIRK currents. (a) Representative GIRK1/2 currents elicited by voltage step to −100 mV for 1 s from a holding potential of 0 mV in the absence and presence of 10 μ
M
fluoxetine. Current responses were recorded in a high-potassium solution containing 96 m
M
K⁺. Arrow indicates the zero current level. (b) Current–voltage relations of 3 m
M
Ba²⁺-sensitive inward currents and 10 μ
M
fluoxetine-sensitive inward currents in oocytes expressing GIRK1/2 channels or GIRK1/4 channels. Current responses were normalized to the 3 m
M
Ba²⁺-sensitive current component measured at a membrane potential of −100 mV. The Ba²⁺-sensitive current components were 2426.9±661.3 nA (n=7) in oocytes expressing GIRK1/2 channels and 1203.3±307.0 nA (n=4) in oocytes expressing GIRK1/4 channels. (c) The percentage inhibition of GIRK channels by fluoxetine over the voltage range of −120 to −20 mV. There was a significant interaction between the fluoxetine effect and the membrane potential effect (P<0.05 for GIRK1/2, n=5–16 for the groups, and P<0.001 for GIRK1/4, n=4 for each group; one-way ANOVA), and then there were significant differences between the effects at −80 or −100 mV and that at −20 mV for GIRK1/2 channels, and between the effects at −60, −80 or −100 mV and that at −20 mV and between the effects at −80 or −100 mV and that at −40 mV for GIRK1/4 channels (P<0.05; Tukey–Kramer *post hoc* test). All values are mean and s.e.m.

**Figure 3**
Concentration–response relations for inhibition of GIRK1/2 channels and GIRK1/4 channels by fluoxetine at three different pH values. The magnitudes of inhibition of GIRK current by fluoxetine were compared with the 3 m
M
Ba²⁺-sensitive current components, which were 1033.3±184.8 nA (n=6, pH 6.0), 741.6±118.0 nA (n=5, pH 7.4) and 822.0±166.3 nA (n=4, pH 9.0) in oocytes expressing GIRK1/2 channels (a), and 625.5±117.4 nA (n=6, pH 6.0), 600.8±268.0 nA (n=5, pH 7.4) and 328.8±114.0 nA (n=4, pH 9.0) in oocytes expressing GIRK1/4 channels (b), respectively. Current responses were measured at a membrane potential of −70 mV in a high-potassium solution. Each point and error bar represents the mean and s.e.m. of the percentage responses obtained. Data points were fitted using a logistic equation.

**Figure 4**
Inhibitory effect of fluoxetine on GIRK channels activated by a G-protein-coupled receptor. (a) In an oocyte coinjected with mRNAs for GIRK1 and GIRK2 channels and XA1 receptor, current responses to adenosine (Ado), Ado in the presence of 10 μ
M
fluoxetine and Ado are shown. The concentration of Ado used was 10 n
M
. Bars show the duration of application. Asterisk indicates the zero current level. (b) Concentration-dependent inhibition of fluoxetine on Ado-induced GIRK currents. I_control is the amplitude of GIRK currents induced by 10 n
M
Ado (407.2±109.0 nA, n=5) and I is the current amplitude in the presence of fluoxetine. Current responses were measured at a membrane potential of −70 mV in a high-potassium solution. Each point and error bar represents the mean and s.e.m. of the relative responses. Data points were fitted using a logistic equation.

**Figure 5**
Comparison of the fluoxetine effects on members of inwardly rectifying potassium channels. (a) Action of fluoxetine on GIRK, ROMK1 and IRK1 channels expressed as homomeric or heteromeric channels in *Xenopus* oocytes. The concentration of fluoxetine used was 100 μ
M
. Current responses were measured at a membrane potential of −70 mV in a high-potassium solution. (b) Distinct concentration-dependent responses to fluoxetine for GIRK2 channels and *weaver* (wv) GIRK2 channels. The magnitudes of inhibition of GIRK current by fluoxetine were compared with the 3 m
M
Ba²⁺-sensitive current components (685.8±183.6 nA, n=5) in oocytes expressing GIRK2 channels, and with the hK current components (2857.8±321.0 nA, n=4) in oocytes expressing wvGIRK2 channels, respectively. Current responses were measured at a membrane potential of −70 mV in a high-potassium solution or a NMDG solution. Each point and error bar represents the mean and s.e.m. of the percentage responses obtained. Data points were fitted using a logistic equation.

**Figure 6**
Inhibitory effect of fluoxetine on the ethanol-induced GIRK currents in *Xenopus* oocytes expressing GIRK1/2 channels. (a) In an oocyte coinjected with GIRK1 and GIRK2 mRNAs, current responses to ethanol (EtOH), EtOH in the presence of 10 μ
M
fluoxetine, EtOH and 3 m
M
Ba²⁺ are shown. The concentration of EtOH used was 100 m
M
. Asterisk indicates the zero current level. Bars show the duration of application. (b) Concentration-dependent inhibition of fluoxetine on EtOH-induced GIRK currents. I_control is the amplitude of GIRK currents induced by 100 m
M
EtOH (340.8±18.1 nA, n=5) and I is the current amplitude in the presence of fluoxetine. Current responses were measured at a membrane potential of −70 mV in a high-potassium solution containing 96 m
M
K⁺. Each point and error bar represents the mean and s.e.m. of the relative responses. Data points were fitted using a logistic equation.

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Inhibition of G protein-activated inwardly rectifying K+ channels by fluoxetine (Prozac) - PubMed