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. 2016 Feb 15;310(4):F311-21.
doi: 10.1152/ajprenal.00436.2015. Epub 2015 Dec 2.

Insulin and IGF-1 activate Kir4.1/5.1 channels in cortical collecting duct principal cells to control basolateral membrane voltage

Oleg Zaika  1 , Oleg Palygin  2 , Viktor Tomilin  3 , Mykola Mamenko  1 , Alexander Staruschenko  2 , Oleh Pochynyuk  4
Affiliations

Insulin and IGF-1 activate Kir4.1/5.1 channels in cortical collecting duct principal cells to control basolateral membrane voltage

Oleg Zaika et al. Am J Physiol Renal Physiol. .

Abstract

Potassium Kir4.1/5.1 channels are abundantly expressed at the basolateral membrane of principal cells in the cortical collecting duct (CCD), where they are thought to modulate transport rates by controlling transepithelial voltage. Insulin and insulin-like growth factor-1 (IGF-1) stimulate apically localized epithelial sodium channels (ENaC) to augment sodium reabsorption in the CCD. However, little is known about their actions on potassium channels localized at the basolateral membrane. In this study, we implemented patch-clamp analysis in freshly isolated murine CCD to assess the effect of these hormones on Kir4.1/5.1 at both single channel and cellular levels. We demonstrated that K(+)-selective conductance via Kir4.1/5.1 is the major contributor to the macroscopic current recorded from the basolateral side in principal cells. Acute treatment with 10 μM amiloride (ENaC blocker), 100 nM tertiapin-Q (TPNQ; ROMK inhibitor), and 100 μM ouabain (Na(+)-K(+)-ATPase blocker) failed to produce a measurable effect on the macroscopic current. In contrast, Kir4.1 inhibitor nortriptyline (100 μM), but not fluoxetine (100 μM), virtually abolished whole cell K(+)-selective conductance. Insulin (100 nM) markedly increased the open probability of Kir4.1/5.1 and nortriptyline-sensitive whole cell current, leading to significant hyperpolarization of the basolateral membrane. Inhibition of the phosphatidylinositol 3-kinase cascade with LY294002 (20 μM) abolished action of insulin on Kir4.1/5.1. IGF-1 had similar stimulatory actions on Kir4.1/5.1-mediated conductance only when applied at a higher (500 nM) concentration and was ineffective at 100 nM. We concluded that both insulin and, to a lesser extent, IGF-1 activate Kir4.1/5.1 channel activity and open probability to hyperpolarize the basolateral membrane, thereby facilitating Na(+) reabsorption in the CCD.

Keywords: PI3-kinase; distal nephron; nortriptyline; sodium reabsorption; transepithelial transport.

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Figures

Fig. 1.
Fig. 1.
Characterization of K+-selective conductance in cortical collecting duct (CCD) principal cells. A: summary graph of resting membrane potentials in cells from freshly isolated CCDs. Two cell populations representing principal (with high negative resting membrane voltage) and intercalated (with low negative resting membrane voltage) are highlighted in circles. B: representative macroscopic currents in individual principal cells in response to voltage steps from −80 to +60 (shown in inset) from the holding potential of −60 mV in the control (black) and upon equimolar substitution of intracellular K+ with Cs+ and in the presence of 30 μM Ba2+ in extracellular medium (light grey). C: current-voltage (I–V) relationships obtained from voltage-step protocols, as shown in B in control (black) and in the presence of Cs+ and Ba2+ (light grey). K+-selective current (grey) was calculated as the difference in the aforementioned conditions. D: summary graph of amplitude of the steady-state current estimated as the absolute current values at the applied voltage of +20 mV. *Significant decrease vs. control.
Fig. 2.
Fig. 2.
Immunolocalization of Kir4.1 and Kir5.1 channels in the renal cortex. A: representative micrograph of double-labeled kidney cortex sections stained for Kir5.1 (red) and aquaporin-2 (AQP2; green) demonstrating colocalization of Kir5.1 with AQP2 on the basolateral membrane of CCD. Staining for Kir5.1 in distal convoluted tubules (DCT) is also apparent. B: representative micrograph of double-labeled kidney cortex sections stained for Kir4.1 (red) and sodium-chloride cotransporter (NCC; green) showing colocalization of Kir4.1 protein with NCC in the DCT. Staining for Kir4.1 in the CCD is also present. G, glomeruli; PT, proximal tubules. Scale bars = 20 μM.
Fig. 3.
Fig. 3.
The basolateral Kir4.1/5.1 channel is the major contributor to the macroscopic current in CCD principal cells. A: representative current traces in response to voltage-step protocol as shown in Fig. 1B in control and after 3-min pretreatment with epithelial sodium channel (ENaC) blocker amiloride, renal outer medullary potassium (ROMK) inhibitor tertiapin-Q (TPNQ), and Na+-K+-ATPase blocker ouabain. Also shown are I–V relationships in control and upon blockade of ENaC (B), ROMK (C), and Na+-K+ ATPase (D). Numbers of individual experiments are also shown.
Fig. 4.
Fig. 4.
Nortriptyline but not fluoxetine drastically decreases the macroscopic K+-selective conductance in CCD principal cells. Shown are average I–V relationships of macroscopic whole cell current in CCD principal cells in control and upon application of Kir4.1 inhibitors 100 μM fluoxetine (A) and 100 μM nortriptyline (B) for 4 min. Numbers of individual experiments are shown.
Fig. 5.
Fig. 5.
Insulin reversibly augments macroscopic currents in CCD principal cells. A: representative whole cell current recordings in response to voltage-step protocol as shown in Fig. 1B in control, after 3-min treatment with insulin (100 nM), and followed washout with regular medium. B: summary graph of changes in amplitude of the steady-state current estimated as the absolute current values at applied voltage of +20 mV in paired experiments similar to that shown in A. *Significant increase vs. control. C: average I–V relationships from individual CCD principal cells in control (black) and after treatment with insulin (light grey). Of note, insulin treatment induces a leftward shift of the reversal potential.
Fig. 6.
Fig. 6.
Inhibition of Kir4.1/5.1 channels with nortriptyline abolishes stimulatory actions of insulin on the macroscopic whole cell current in CCD principal cells. A: summary graph of changes in amplitude of the steady-state current estimated as the absolute current values at applied voltage of +20 mV in control, after pretreatment with 100 μM nortriptyline, simultaneous application of 100 nM insulin and 100 μM nortriptyline, then washout with control media. *Significant decrease vs. control. B: average I–V relationships from individual CCD principal cells pretreated with 100 μM nortriptyline at baseline and in the presence of insulin (light grey).
Fig. 7.
Fig. 7.
Insulin acutely increases open probability (Po) of Kir4.1/5.1 channel via activation of PI3-kinase. A: representative continuous current trace from a cell-attached patch monitoring activity of basolateral Kir4.1/5.1 channels in CCD principal cells in control, after application of 100 nM insulin, and followed washout with the control medium. The patch was clamped to −Vp = −40 mV. Insulin application time is shown by the bar on top. Areas 1 and 2 are shown below at an expanded timescale. Insulin treatment mildly increases the unitary current amplitude, indicative of basolateral membrane hyperpolarization. B: summary graph of changes in Kir4.1/5.1 Po upon treatment with 100 nM insulin from paired patch-clamp experiments similar to that shown in A. *Significant increase vs. control. C: summary graph of changes in Kir4.1/5.1 Po in paired cell-attached patch-clamp experiments in control, upon treatment with phosphatidylinositol 3-kinase (PI3)-K inhibitor LY294002 (20 μM), insulin (100 nM) in the continued presence of the inhibitor, and following washout.
Fig. 8.
Fig. 8.
High levels of IGF-1 stimulate macroscopic currents in CCD principal cells. A: representative whole cell current recordings in response to voltage-step protocol as shown in Fig. 1B in control, after 3 min treatment with IGF-1 (500 nM), and followed washout with regular medium. B: summary graph of changes in amplitude of the steady-state current estimated as the absolute current values at applied voltage of +20 mV in paired experiments similar to that shown in A. *Significant increase vs. control. C: average I–V relationships from individual CCD principal cells in control (black) and after treatment with IGF-1 (light grey). Similarly to the effect of insulin in Fig. 5C, IGF-1 application shifts the reversal potential to more negative values.
Fig. 9.
Fig. 9.
High concentration of IGF-1 augments Kir4.1/5.1 single-channel activity in a PI3-kinase dependent manner. A: representative continuous current trace from a cell-attached patch monitoring activity of basolateral Kir4.1/5.1 channels in CCD principal cells in control, after application of 500 nM IGF-1, and followed washout with the control medium. The patch was clamped to −Vp = −40 mV. IGF-1 application time is shown with the bar on top. Areas 1 and 2 are shown below at an expanded timescale. IGF-1 treatment mildly increases the unitary current amplitude, indicative of basolateral membrane hyperpolarization. B: summary graph of changes in Kir4.1/5.1 Po upon treatment with 500 nM IGF-1 from paired patch-clamp experiments similar to that shown in A. *Significant increase vs. control. C: summary graph of changes in Kir4.1/5.1 Po in paired cell-attached patch-clamp experiments in control, upon treatment with PI3-K inhibitor LY294002 (20 μM), IGF-1 (500 nM) in the continued presence of the inhibitor, and following washout.
Fig. 10.
Fig. 10.
Insulin and IGF-1 hyperpolarize basolateral membrane in CCD principal cells. Representative continuous voltage traces monitoring basolateral membrane potential in individual cells in control and after application of 100 nM insulin (A) and 500 nM IGF-1 (B). Short applications of 50 mM KCl were used to assess cell viability. Drug application times are shown with respective bars on the top. C: summary graph of the resting basolateral membrane potential in control and after acute application of insulin and IGF-1. *Significant increase vs. control.
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