Ca2+-Dependent Inhibition of Inwardly Rectifying K+ channel in Opossum Kidney Cells.

Abstract

It has been demonstrated in both mammalian and amphibian proximal tubule cells with the patch-clamp and microelectrode techniques that the inwardly rectifying pH-sensitive and ATP-regulated K ϩ channel with a conductance of about 50 pS mediate the membrane potential of proximal tubule cells [1][2][3]. We have demonstrated that in cultured opossum kidney proximal tubule (OKP) cells, the inwardly rectifying pH-sensitive K ϩ channel with a conductance of about 70-90 pS is regulated by ATP, protein kinase A (PKA), and inhibited by Ba 2ϩ or glybenclamide, a specific inhibitor of sulfonylurea receptor [4][5][6]. Besides electrophysiological studies, the cloning and reconstitution studies demonstrated that ATP-sensitive K ϩ channels are heteromultimers of inwardly rectifying K ϩ channel subunits (Kir6.x) and sulfonylurea receptors (SUR) [7]. Moreover, it has also been indicated with the histochemical studies of the inwardly rectifying ATP-sensitive K ϩ channel (Kir6.1) that Kir6.1 with a conductance of 70 pS is ubiquitously Japanese Journal of Physiology, 51, 371-380, 2001 Key words: proximal tubule cell, inwardly rectifying K ؉ channel, PKC, CaMK II, calcineurin. Abstract:The effect of intracellular Ca 2ϩ on the activity of the inwardly rectifying ATP-regulated K ϩ channel with an inward conductance of about 90 pS was examined by using the patchclamp technique in opossum kidney proximal tubule (OKP) cells. The activity of the inwardly rectifying K ϩ channel rapidly declined with an application of ionomycin (1 M) in the presence of 10 Ϫ6 M Ca 2ϩ in cell-attached patches. The application of 10 M phorbor-12-myristate-acetate (PMA) with 10 Ϫ6 M Ca 2ϩ reduced the K ϩ channel activity. Although the channel activity was not influenced by an increase of bath Ca 2ϩ from 10 Ϫ7.5to 10 Ϫ6 M, the activity was inhibited by protein kinase C (PKC, 1 U/ml) with 10 Ϫ6 M Ca 2ϩ in insideout patches. The inhibitory effect of Ca 2ϩ with ionomycin on the channel activity was diminished by the pretreatment with a specific PKC inhibitor, GF 109203X (5 M), in cell-attached patches. By contrast, the application of Ca 2ϩ /calmodulin kinase II (CaMK II, 300 pM) dramatically increased this channel activity in inside-out patches. In cellattached patches, the addition of both GF 109203X and cyclospolin A (5 M), a potent inhibitor of protein phosphatase 2B (calcineurin), instead stimulated the K ϩ channel activity with ionomycin and 10 Ϫ6 M Ca 2ϩ. The addition of protein phosphatase 2B (calcineurin) (2 U/ml) to the bath with calmodulin (1 M) and Ni 2ϩ (10 M) to stimulate calcineurin inhibited the channel activity in inside-out patches. Furthermore, the inhibitory effect of PKC or calcineurin on this channel activity was abolished by a removal of Ca

Constitutive Activity of Inwardly Rectifying K+ Channel at Physiological [Ca]i Is Mediated by Ca2+/CaMK II Pathway in Opossum Kidney Proximal Tubule Cells

“…The two main groups of Ca 2+ -dependent protein kinases are (i) protein kinase C (PKC) and (ii) Ca 2+ /calmodulindependent protein kinase II (CaMK II). Previously, we studied the effect of PKC on this K + channel and demonstrated that PKC inhibits the inwardly rectifying K + channels at the intracellular Ca 2+ concentration ([Ca] i ) over the physiological range, ~10 -6.5 M [9]. Furthermore, we reported that CaMK II-mediated phosphorylation contributes to the activation of inwardly rectifying K + channels in OKPCs [9].…”

“…Previously, we studied the effect of PKC on this K + channel and demonstrated that PKC inhibits the inwardly rectifying K + channels at the intracellular Ca 2+ concentration ([Ca] i ) over the physiological range, ~10 -6.5 M [9]. Furthermore, we reported that CaMK II-mediated phosphorylation contributes to the activation of inwardly rectifying K + channels in OKPCs [9].Although the effect of Ca 2+ -dependent proteins, such as the constitutive form of nitric oxide synthase (cNOS) [13,14] In this study, therefore, we examined the effect of a low [Ca] i on the inwardly rectifying K + channels in OKPCs, …”

“…It has also been shown that Ca 2+ -dependent signal transduction systems regulate the activity of the inwardly rectifying K + channel in the proximal tubule cells [9,10]. Ca 2+ -dependent serine/threonine protein kinases are major components of the Ca 2+ -dependent signal transduction system [11,12], and these kinases are known to regulate the activity of several ion channels, carriers, and pumps, thereby modulating transmembrane electrolyte transport.…”

“…Although the effect of Ca 2+ -dependent proteins, such as the constitutive form of nitric oxide synthase (cNOS) [13,14] or PKC [9], on K + channel activity has been studied at a high cytosolic Ca 2+ concentration, it has not been fully examined at a physiological or low cytosolic Ca 2+ concentration. Furthermore, our previous experiments left the following question open [9]: does CaMK II activate this K + channel at physiological [Ca] i , such as ~10 -7 M? In this study, therefore, we examined the effect of a low [Ca] i on the inwardly rectifying K + channels in OKPCs, hematocrit capillaries (Nichiden Rika Glass, Hyogo, Japan).…”

“…Furthermore, our previous experiments left the following question open [9]: does CaMK II activate this K + channel at physiological [Ca] i , such as ~10 -7 M? In this study, therefore, we examined the effect of a low [Ca] i on the inwardly rectifying K + channels in OKPCs, hematocrit capillaries (Nichiden Rika Glass, Hyogo, Japan). The filling solution of the patch pipettes for singlechannel recordings contained 145 mM K + solution (10 -7.5 M free Ca 2+ ).…”