The A kinase anchoring protein is required for mediating the effect of protein kinase A on ROMK1 channels

Abstract

In the present study, we have used the two-electrode voltage-clamp and patch-clamp techniques to study the effects of forskolin and cAMP on the ROMK1 channels, which are believed to be the native K+ secretory channels in the kidney. Addition of 1 microM forskolin or 100 microM 8-bromo-cAMP, within 10 min, has no significant effect on the current of ROMK1 channels expressed in Xenopus oocytes. In contrast, application of 1 microM forskolin, within 3 min, significantly increased whole-cell K+ current by 35%, when ROMK1 channels were coexpressed with the A kinase anchoring protein AKAP79, which was cloned from neuronal tissue. Two lines of evidence indicate that the effect of forskolin is mediated by a cAMP-dependent pathway: (i) Addition of 100 microM 8-bromo-cAMP mimics the effect of forskolin and (ii) the effect of forskolin and cAMP is not additive. That AKAP is required for the effect of cAMP is further supported by experiments in which addition of ATP (100 microM) and cAMP (100 microM) restored the activity of run-down ROMK1 channels in inside-out patches in oocytes that coexpressed ROMK1 and AKAP79 but not in those that expressed ROMK1 alone. Moreover, when we used RII, the regulatory subunit of type II protein kinase A, in an overlay assay, we identified a RII-binding protein in membranes obtained from the kidney cortex but not in membranes from oocytes. This suggests that the insensitivity of ROMK1 channels to forskolin and cAMP is due to the absence of AKAPs. We conclude that AKAP may be a critical component that mediates the effect of protein kinase A on the ROMK channels in the kidney.

Figure 1

(A) Representative trace showing the effects of 1 μM forskolin on the Ba²⁺-sensitive channel current measured by the two-electrode voltage-clamp technique. The oocytes were clamped at −60 mV. The dotted line indicates the closed current. Addition of forskolin and Ba²⁺ is indicated by arrows. (B) Effect of 1 μM forskolin and 100 μM 8-Br-cAMP (cAMP) on the Ba²⁺-sensitive K⁺ current.

Figure 2

Autoradiograph showing the results of ³²P-labeled RII overlay assay. The RII-binding protein with a molecular mass of 100-120 kDa in brain and kidney membranes (cortex) is shown.

Figure 3

Representative trace showing the effect of 1 μM forskolin on the Ba²⁺-sensitive K⁺ current when oocytes were injected with cRNAs encoding the AKAP79 and ROMK1 channel. The dotted line indicates the closed level of channel current and arrows indicate where the agents were added to the bath.

Figure 4

Effect of 1 μM forskolin on ROMK1 channel current with coexpression of AKAP79 (○) and without AKAP79 (•). Asterisks indicate that data were significantly different from the control value.

Figure 5

Effects of 1 μM forskolin and 100 μM 8-Br-cAMP (cAMP) on ROMK1 channel current when oocytes were injected with cRNAs for AKAP79 and ROMK1 channels. Asterisks indicate that data are significantly different from the control value.

Figure 6

Channel recording showing the effects of 100 μM MgATP and 100 μM cAMP on ROMK1 channel activity when oocytes were injected with ROMK1 channel alone (Upper) and injected with both ROMK1 channel and AKAP79 (Lower). The closed level of channel current (C) is indicated, and the number of channels in the patch is also indicated. Experiments were performed in inside-out patches and the cell membrane potential was −30 mV. The gap in the trace is 60 s.

Figure 7

Schema illustrating the role of AKAP in mediating the PKA stimulation of ROMK1-like channels in the kidney. Stimulation of the receptor’s (R) coupling to the G-protein (G_s) activates adenylate cyclase (AC), which increases cAMP production. cAMP can consequently stimulate PKA, which binds to the cell membrane through the AKAP. Accordingly, the ROMK channel is phosphorylated (P).