PKC and AMPK regulation of Kv1.5 potassium channels

Abstract

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K(+) current (IKur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells. By confocal microscopy combined with electrophysiology we demonstrate that PKC activation reduces Kv1.5 current, through a decrease in membrane expressed channels. AMPK activation was found to decrease the membrane expression in MDCK cells, but not in HL-1 cells and was furthermore shown to be dependent on co-expression of Nedd4-2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems.

Figure 1.

Kv1.5 surface expression is reduced in response to cell polarization, to PKC activation and to AMPK activation in MDCK cells. (A) MDCK cells transiently co-transfected with DsRed-ER and Kv1.5 cDNA and subjected to a calcium switch assay (see Materials and Methods). At different time points after initiation of the calcium switch, coverslips were fixed and stained for Kv1.5. Illustrated are confocal horizontal scans of the MDCK cells before the initiation of the calcium switch (t =0h, cells grown in low calcium medium) and 30 min and 3h after the initiation of the polarization process following addition of calcium containing medium. As illustrated, surface expressed Kv1.5 channels disappear from the membrane during the 3 hour calcium switch. (B and C) MDCK cells transiently co-transfected with DsRed-ER and Kv1.5 and grown in low calcium media. The cells were treated with the PKC activator PMA (100 nM) (B) or the 2 AMPK activators AICAR (500 μM) and PT1 (200 μM) (C) for up to 6 hours. As illustrated, both activation of PKC and AMPK lead to disappearance of surface expressed Kv1.5 channels. Cytoskeletal actin staining was used as a membrane marker. Scalebar,10 μm.

Figure 2.

PKC but not AMPK activation reduces Kv1.5 surface expression in HL-1 cells. (A and B) Confocal scans of HL-1 cells transiently transfected with Kv1.5 cDNA and treated with PMA (100 nM), AICAR (1 mM) or PT1 (200 μM). As illustrated, surface expression of Kv1.5 channels was reduced in response to PKC activation (A). This response was not observed with either of the 2 AMPK activators (B). Cytoskeletal actin staining was used as a marker for the membrane. Scalebar, 10 μm. (C) Representative traces of Kv1.5 currents recorded in HL-1 cells before and after treatment with PMA. Protocol shown in the insert. (D) Current-voltage relationship of currents recorded in Kv1.5 transfected and untransfected HL-1 cells. PMA significantly down regulated the current level by 74%. Numbers of cells are: Kv1.5 (n=12); Kv1.5+PMC (n=13); untransfected (n=8); untransfected+PMA (n=10).

Figure 3.

Two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing Kv1.5 +/− Nedd4–2 following PKC and AMPK activation. (A-D) Representative Kv1.5 current recordings following a step protocol. (E) PKC activation by PMA resulted in a drastic Kv1.5 current reduction both in the presence and absence of co-expressed Nedd4–2. (E) Similar experiments were conducted using the AMPK activator ZMP (100 nM). Contrary to PKC activation, AMPK activation did not affect Kv1.5 current levels when Nedd4–2 was not co-expressed. However, when Nedd4–2 was co-injected, leading to a down-regulation of Kv1.5 surface current, ZMP induced a further current reduction. The number of cells in each group was (n>10).

Figure 4.

Schematic illustration of the PKC and AMPK pathways resulting in Kv1.5 downregulation. The polarization of MDCK cells triggers activation of PKC and AMPK kinases. PKC activation can be mimicked by PMA. We suggest this activation of PKC can impact Kv1.5 channels through 2 pathways, depending on which the cell system used. This might be through AMPK activation (that can be mimicked by PT1, AICAR and ZMP), which will activate Nedd4 ubiquitylating enzymes or by another so far undisclosed mechanism. Activation of both pathways results in a reduced Kv1.5 surface expression.