Insulin and IGF - 1 activate Kir 4 . 1 / 5 . 1 channels in cortical 1 collecting duct principal cells to control basolateral membrane

25 26 Potassium Kir4.1/5.1 channels are abundantly expressed at the basolateral membrane of 27 principal cells in the cortical collecting duct (CCD) where they are thought to modulate 28 transport rates by controlling trans-epithelial voltage. Insulin and insulin-like growth factor-1 29 (IGF-1) stimulate apically localized ENaC to augment sodium reabsorption in the CCD. 30 However, little is known about their actions on potassium channels localized at the basolateral 31 membrane. In this study, we implemented patch clamp analysis in freshly isolated murine 32 CCD to assess the effect of these hormones on Kir4.1/5.1 at both single channel and cellular 33 levels. We demonstrated that K-selective conductance via Kir4.1/5.1 is the major contributor 34 to the macroscopic current recorded from the basolateral side in principal cells. Acute 35 treatment with 10 μM amiloride (ENaC blocker), 100 nM Tertiapin-Q (TPNQ, ROMK inhibitor), 36 and 100 μM ouabain (Na-K ATPase blocker) failed to produce a measurable effect on the 37 macroscopic current. In contrast, Kir4.1 inhibitor nortriptyline (100 μM), but not fluoxetine (100 38 μM) virtually abolished whole cell K-selective conductance. Insulin (100 nM) markedly 39 increased open probability of Kir4.1/5.1 and nortriptyline-sensitive whole cell current leading to 40 significant hyperpolarization of the basolateral membrane. Inhibition of PI3-kinase cascade 41 with LY294002 (20 μM) abolished action of insulin on Kir4.1/5.1. IGF-1 had similar stimulatory 42 actions on Kir4.1/5.1-mediated conductance only when applied at a higher (500 nM) 43 concentration and was ineffective at 100 nM. We concluded that both insulin and, to a lesser 44 extent, IGF-1 activate Kir4.1/5.1 channels activity and open probability to hyperpolarize the 45 basolateral membrane thereby facilitating Na reabsorption in the CCD. 46 47