Protein kinase G activates inwardly rectifying K⁺channel in cultured human proximal tubule cells

Abstract

An ATP-regulated inwardly rectifying K(+) channel, whose activity is enhanced by PKA, is present in the plasma membrane of cultured human proximal tubule cells. In this study, we investigated the effects of PKG on this K(+) channel, using the patch-clamp technique. In cell-attached patches, bath application of a membrane-permeant cGMP analog, 8-bromoguanosine 3',5'-monophosphate (8-BrcGMP; 100 microM), stimulated channel activity, whereas application of a PKG-specific inhibitor, KT-5823 (1 microM), reduced the activity. Channel activation induced by 8-BrcGMP was observed even in the presence of a PKA-specific inhibitor, KT-5720 (500 nM), which was abolished by KT-5823. Direct effects of cGMP and PKG were examined with inside-out patches in the presence of 1 mM MgATP. Although cytoplasmic cGMP (100 microM) alone had little effect on channel activity, subsequent addition of PKG (500 U/ml) enhanced it. Furthermore, bath application of atrial natriuretic peptide (ANP; 20 nM) in cell-attached patches stimulated channel activity, which was blocked by KT-5823. In conclusion, cGMP/PKG-dependent processes participate in activating the ATP-regulated K(+) channel and producing the stimulatory effect of ANP on channel activity.

Regulation of an inwardly rectifying K+channel by nitric oxide in cultured human proximal tubule cells

“…4C). This is in good agreement with the previous observation that 8-BrcGMP restored the suppressed channel activity by KT-5720 (28). Summarized data are shown in Fig.…”

“…The inward slope conductance, which was measured between Ϫ90 and Ϫ30 mV of ϪV p , was 42.8 Ϯ 1.1 pS, whereas the outward slope conductance between ϩ30 and ϩ90 mV was 8.5 Ϯ 2.7 pS. These measurements almost fit with the previous results obtained from inside-out patches under the symmetrical high-K ϩ condition (28,29). When the bath was replaced with the control solution, the current-voltage curve shifted to the right with the change in reversal potential from ϩ10 to ϩ75 mV (Fig.…”

“…28). We further confirmed that atrial natriuretic peptide (ANP), which stimulates ANP receptorcoupled guanylate cyclase and hence cGMP synthesis, elevated channel activity through PKG-mediated phosphorylation (28). Such characteristics are similar to those of the K ϩ channel identified in opossum kidney proximal tubule cells (17,18).…”

“…We previously reported that hANP increased the activity of this K ϩ channel through cGMP/PKG-mediated processes (28). Although ANP is believed to act on its target cells through activation of particulate guanylate cyclase, a few reports suggest that ANP stimulated NO release from human proximal tubular cells (5,24).…”

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 activation of nNOS and eNOS, both of which require the elevation of [Ca] i , leads to the activation of a cGMP-mediated phosphorylation process [31]. Several reports have demonstrated that cGMP-mediated phsophorylation regulates inwardly rectifying K + channels in proximal tubule cells [6,32,33]. In the present study, however, the application of L-NNA or L-NAME to the bath did not affect the activity of inwardly rectifying K + channels ( Fig.…”