Sphingosine-1-phosphate (S1P) is an endogenous agonist for a family of five G protein-coupled receptors (S1P 1-5 ) involved in cell proliferation, cardiovascular development and lymphocyte trafficking. The sphingolipid drug FTY720 displays structural similarity to S1P and efficacy as an immunosuppressant in models of autoimmune disease and in solid organ transplantation. While FTY720 is well-tolerated in humans, it produces a transient reduction of heart rate (HR). As S1P activates the cardiac G protein-gated potassium channel I KACh , we speculated that the FTY720-induced HR reduction reflects I KACh activation. We examined FTY720 effects on atrial myocytes from wild-type and I KACh -deficient mice. In wild-type myocytes, the active phosphate metabolite of FTY720 (FTY720-P) induced single channel activity with conductance, open time, GTP sensitivity and rectification identical to that of I KACh . In whole-cell recordings, FTY720-P evoked an inwardly rectifying potassium current in ∼90% of myocytes responding to acetylcholine. Comparable channel activity was never observed in myocytes from I KAChdeficient mice. In wild-type mice, acute FTY720 administration produced a dose-dependent, robust HR reduction. In contrast, the HR reduction induced by FTY720 in I KACh -deficient mice was blunted. We conclude that the effect of acute FTY720 administration on HR is mediated primarily by I KACh activation.

The muscarinic-gated atrial potassium (I KACh ) channel contributes to the heart rate decrease triggered by the parasympathetic nervous system. I KACh is a heteromultimeric complex formed by Kir3.1 and Kir3.4 subunits, although Kir3.4 homomultimers have also been proposed to contribute to this conductance. While Kir3.4 homomultimers evince many properties of I KACh , the contribution of Kir3.1 to I KACh is less well understood. Here, we explored the significance of Kir3.1 using knock-out mice. Kir3.1 knock-out mice were viable and appeared normal. The loss of Kir3.1 did not affect the level of atrial Kir3.4 protein but was correlated with a loss of carbachol-induced current in atrial myocytes. Low level channel activity resembling recombinant Kir3.4 homomultimers was observed in 40% of the cellattached patches from Kir3.1 knock-out myocytes. Channel activity typically ran down quickly, however, and was not recovered in the inside-out configuration despite the addition of GTP and ATP to the bath. Both Kir3.1 knock-out and Kir3.4 knock-out mice exhibited mild resting tachycardias and blunted responses to pharmacological manipulation intended to activate I KACh . We conclude that Kir3.1 confers properties to I KACh that enhance channel activity and that Kir3.4 homomultimers do not contribute significantly to the muscarinic-gated potassium current.

Our aim was to determine whether G protein-gated potassium (Kir3) channels contribute to thermonociception and morphine analgesia. Western blotting was used to probe for the presence of Kir3.1, Kir3.2, Kir3.3, and Kir3.4 subunits in the mouse brain and spinal cord. Hot-plate paw-lick latencies for wild-type, Kir3.2 knockout, Kir3.3 knockout, and Kir3.4 knockout mice were measured at 52 degrees C and 55 degrees C, following the s.c. injection of either saline or 10 mg/kg morphine. Paw-lick latencies for Kir3.4 knockout mice were similar to those of wild-type mice, consistent with the restricted expression pattern of Kir3.4 subunit in the mouse brain. In contrast, Kir3.2 knockout and Kir3.3 knockout mice displayed hyperalgesia at both temperatures tested, and both Kir3.2 knockout and Kir3.3 knockout mice displayed shorter paw-lick latencies following morphine administration, with Kir3.2 knockout mice exhibiting the more dramatic phenotype. Kir3.2/Kir3.3 double knockout mice displayed a greater degree of hyperalgesia than either the Kir3.2 knockout or Kir3.3 knockout mice, while performing similarly to Kir3.2 knockout mice following morphine administration. We conclude that G protein-gated potassium channels containing Kir3.2 and/or Kir3.3 play a significant role in responses to moderate thermal stimuli. Furthermore, the activation of Kir3 channels containing the Kir3.2 subunit contributes to the analgesia evoked by a moderate dose of morphine. As such, receptor-independent Kir3 channel agonists may represent a novel and selective class of analgesic agent.