Vanishing Act: PKC-dependent internalization of K_ATP channels

Potassium channels play an important role in the regulation of vascular smooth muscle tone and thus contribute to the regulation of blood pressure, blood flow and microvascular exchange ¹. These channels importantly participate in the determination of vascular smooth muscle cell (VSMC) membrane potential ^{1, 2}, which, in turn, controls Ca²⁺ influx through voltage-gated Ca²⁺ channels ^{1, 2} and has been implicated in the control of Ca²⁺ release and Ca²⁺ sensitivity of VSMC ¹. VSMC express a diverse array of K⁺ channels that contribute to the regulation of VSMC function ¹ including one or more types of ATP-sensitive K⁺ (K_ATP) channels ^{2, 3}.

K_ATP channels consist of a tetramer of α-pore forming subunits from the KIR 6.X family of inwardly rectifying K⁺ channels, along with complimentary sulfonylurea receptor (SUR) subunits that are members of the ATP-binding cassette (ABC) family of proteins ². The SUR subunits are essential for normal trafficking of K_ATP channels, modulate channel function and are the binding site for sulfonylurea antagonists of these channels, such as glibenclamide ². Vascular smooth muscle K_ATP channels appear to be composed of KIR6.1 and SUR2B subunits ², although some VSMC also may express K_ATP channels composed of KIR6.2/SUR2B ³.

As originally described, K_ATP channels open during hypoxia or ischemic conditions when cellular ATP levels fall, decreasing cell excitability protecting energy-limited cells ². However, both in vitro and in vivo studies suggest that VSMC K_ATP channels may be open under resting conditions and contribute to the regulation of VSMC membrane potential and vascular tone ^{1, 2}. Importantly, the activity of K_ATP channels can be modulated by a number of physiologically relevant vasoactive substances and conditions. As their name implies, K_ATP channels may be activated by decreases in intracellular ATP and appear be important sensors of the metabolic status of cells, opening during ischemic or hypoxic conditions to promote vasodilation and an increase in blood flow and oxygen delivery ^{1, 2}. K_ATP channels also are modulated by a plethora of additional intracellular signals including ADP, H⁺ and Ca^{2+ 1, 2}. Cyclic AMP, acting through protein kinase A (PKA), activates VSMC K_ATP channels such that vasodilators including isoproterenol, adenosine, PGI₂, and calcitonin-gene-related-peptide (CGRP) act, in part, through these channels ^{1, 2}. In contrast, vasoconstrictors that act through G-protein coupled receptors such as norepinephrine, phenylephrine, serotonin, histamine, neuropeptide Y (NPY), endothelin, vasopressin and angiotensin-II all inhibit VSMC K_ATP channels ^{1, 2}. Vasoconstrictor-induced inhibition of K_ATP channels results from at least three mechanisms: Ca²⁺-dependent activation of the phosphatase, calcineurin (protein phosphatase 2B or protein phosphatase 3) ⁴, G_i/o-mediated inhibition of constituitive adenylate cyclase activity ⁵ and activation of protein kinase C (PKC) ^{3, 5, 6} (see Figure 1). The study by Jiao et al. ⁷ in this issue of Hypertension confirms and extends these studies demonstrating an important role for PKCε in inhibition of K_ATP channel currents in both HEK cells and VSMC by phorbol esters and angiotensin II.

Figure 1.

Mechanisms of inhibition of K_ATP channel currents in vascular smooth muscle cells. Schematic of the signaling pathways involved in inhibition of K_ATP channels by vasoconstrictors such as angiotensin II (AngII). As highlighted in the text, AngII appears to inhibit currents through K_ATP channels (I_KATP) by several mechanisms: Internalization of K_ATP channels composed of KIR6.1/SUR2B subunits via a mechanism involving PKCε, inhibition of steady-state adenylate cyclase (AC) activity, and Ca²⁺-dependent activation of the phosphatase, calcineurin (CaN), although the molecular mechanisms by which these latter two pathways inhibit K_ATP channel function remains to be established (indicated by “?”). AT1R – AngII type 1 receptor; G_q/11 – Phopholipase C (PLC) coupled G-protein; G_i/o – inhibitory G-protein; DAG – diacylglycerol; IP₃ – inositol 1,4,5-trisphosphate; PKA – protein kinase A; I_VGCC – current through voltage-gated Ca²⁺ channels; [Ca²⁺]_in – intracellular Ca²⁺ concentration. See text for more information.

PKC has been implicated in vasoconstrictor-induced inhibition of K_ATP channels for more than a decade (see reference ⁵ for older literature). Prior studies identified PKCε as an important isoform in VSMC, and indicated that targeting of K_ATP channels and PKCε to caveolae was important in this interaction ⁶. However, the mechanism by which PKCε inhibits K_ATP channels remained unclear. Jiao et al., ⁷ present data showing that PKC-induced inhibition of K_ATP channels, both in HEK cells and native VSMCs, involves caveolin-dependent internalization of the channels (Figure 1). They showed that PKC-dependent inhibition of KIR 6.1/SUR2B channels expressed in HEK cells, as well as K_ATP channels expressed in dermal VSMCs was associated with redistribution of the channels from the plasma membrane into the cytosol, and that both inhibition of K_ATP channel currents and internalization were reduced by expression of a dominant negative form of dynamin. Disruption of caveolae by removal of membrane cholesterol with methyl-β-cyclodextrin prevented, whereas overexpression of caveolin-1 potentiated the inhibitory effects of PKC in HEK cells. Similarly, in VSMCs, Jiao et al., ⁷ showed that angiotensin II-induced inhibition of pinacidil-stimulated K_ATP currents and stimulation of K_ATP channel internalization could be blunted by PKC antagonists, expression of a dominant negative form of dynamin or siRNA knockdown of caveolin-1. These experiments show that rapid, PKC-dependent internalization of VSMC K_ATP channels may underlie PKC-dependent inhibition of K_ATP channel currents adding to our understanding of the regulation of these channels by vasoactive substances. However, a number of questions remain to be answered.

First, how does PKC lead to K_ATP channel internalization? Studies of KIR 6.2/SUR1/2A-based K_ATP channels also have demonstrated PKC-dependent inhibition of K_ATP channel currents (which occurs after an initial stimulation in this channel form) involving PKC-stimulated channel endocytosis ⁸. These studies further showed that a di-leucine motif in the C-terminus of KIR 6.2 was essential for PKC-induced internalization, but that PKC-mediated phosphorylation of KIR 6.2 was not involved in the process. These data suggest that PKC-dependent phosphorylation of some other target participates in channel endocytosis. Recent studies of KIR 6.1/SUR2B channels expressed in HEK cells indicate that this may not be the case for VSMC K_ATP channels ⁹. In this system, phosphorylation of several serine residues in the C-terminus of KIR 6.1 appeared essential for PKC-mediated inhibition of K_ATP channel currents. However, it is worthy to note that channel internalization was not examined in this study and should be investigated in the future. Thus, the molecular details of how activation of PKCε leads to internalization of VSMC K_ATP channels remains to be established.

Second, what is the fate of internalized VSMC K_ATP channels? PKC-dependent internalization of KIR 6.2/SUR1/2A-based K_ATP channels leads to appearance of some of these channels in late endosomes/lysosomes along with Rab 7 ⁸, a marker of clathrin-mediated endocytosis, suggesting that the channels were fated for degradation. Whether this also is true for KIR 6.1/SUR2B channels expressed in VSMC will require further investigation.

Third, is channel internalization the only means by which activated PKC inhibits K_ATP channels? Studies of native VSMC K_ATP channels ³ have shown that exogenous PKC reversibly inhibits K_ATP channel activity by increasing the interburst interval. As all recordings were performed with multiple channels in the patches, such behavior could have resulted from PKC-stimulated channel internalization and a decrease in the number of channels per patch, with recovery of channel activity by rapid reinsertion of channels into the membrane patch upon washout of the PKC. Recent studies of Kv1.5 channel recycling in atrial myocytes demonstrates recovery of internalized channels with a half-time for recovery on the order of 29 min ¹⁰. This is considerably slower than the 5 min cited for recovery of VSMC K_ATP channel currents in inside-out patches after washout of PKC ³. Thus, it may be that PKC has multiple actions, affecting both gating and trafficking of VSMC K_ATP channels, perhaps independently. Additional studies will be required to resolve this issue.

Finally, as noted above, vasoconstrictors, such as angiotensin II, also can inhibit K_ATP channels by at least two additional mechanisms: receptor-mediated inhibition of constituitive adenylate cyclase activity ⁵ and Ca²⁺-dependent activation of protein phosphatase 2B (calcineurin) ⁴. Whether these pathways also involve modulation of K_ATP channel trafficking remains to be established.

Thus, while the studies of Jiao and colleagues ⁷ move our understanding of the mechanism by which PKC inhibits VSMC K_ATP channels forward, additional studies will be required to define the molecular details of PKC-induced K_ATP channel internalization, and critically, the importance of this process in the maintenance of cardiovascular homeostasis in health and disease.