Protein Kinase C–Dependent Internalization of Adenosine 5′-Triphosphate–Sensitive K+ 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+ channels1,2 and has been implicated in the control of Ca2+ release and Ca2+ sensitivity of VSMCs.1 VSMCs express a diverse array of K+ channels that contribute to the regulation of VSMC function,1 including ≥1 type of vascular ATP-sensitive K+ (KATP) channels.2,3
KATP channels consist of a tetramer of α-pore–forming subunits from the KIR6.X family of inwardly rectifying K+ channels, along with complimentary sulfonylurea receptor (SUR) subunits that are members of the ATP-binding cassette family of proteins.2 The SUR subunits are essential for normal trafficking of KATP channels, modulate channel function, and are the binding sites for sulfonylurea antagonists of these channels, such as glibenclamide.2 Vascular smooth muscle KATP channels appear to be composed of KIR6.1 and SUR2B subunits,2 although some VSMCs may also express KATP channels composed of KIR6.2/SUR2B.3
As originally described, KATP channels open during hypoxia or ischemic conditions when cellular ATP levels fall, decreasing cell excitability and protecting energy-limited cells.2 However, both in vitro and in vivo studies suggest that VSMC KATP 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 KATP channels can be modulated by a number of physiologically relevant vasoactive substances and conditions. As their name implies, KATP 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 KATP channels also are modulated by a plethora of additional intracellular signals including ADP, H+, and Ca2+.1,2 cAMP, acting through protein kinase A, activates VSMC KATP channels such that vasodilators including isoproterenol, adenosine, prostaglandin I2, and calcitonin-gene-related-peptide 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, endothelin, vasopressin, and angiotensin II all inhibit VSMC KATP channels.1,2 Vasoconstrictor-induced inhibition of KATP channels results from ≥3 mechanisms: Ca2+-dependent activation of the phosphatase, calcineurin (protein phosphatase 2B or protein phosphatase 3),4 Gi/o-mediated inhibition of constitutive adenylate cyclase activity,5 and activation of protein kinase C (PKC)3,5,6 (see the Figure). The study by Jiao et al7 in this issue of Hypertension confirms and extends these studies, demonstrating an important role for PKC-ε in the inhibition of KATP channel currents in both human embryonic kidney (HEK) cells and VSMCs by phorbol esters and angiotensin II.
PKC has been implicated in vasoconstrictor-induced inhibition of KATP channels for more than a decade (see Reference 5 for older literature). Previous studies identified PKC-ε as an important isoform in VSMCs and indicated that targeting of KATP channels and PKC-ε to caveolae was essential in this interaction.6 However, the mechanism by which PKC-ε inhibits KATP channels remained unclear. Jiao et al7 present data showing that PKC-induced inhibition of KATP channels, both in HEK cells and native VSMCs, involves caveolin-dependent internalization of the channels (Figure). They showed that PKC-dependent inhibition of KIR 6.1/SUR2B channels expressed in HEK cells, as well as KATP channels expressed in dermal VSMCs, was associated with redistribution of the channels from the plasma membrane into the cytosol and that both inhibition of KATP 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 al7 showed that angiotensin II–induced inhibition of pinacidil-stimulated KATP currents and stimulation of KATP 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 KATP channels may underlie PKC-dependent inhibition of KATP 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 KATP channel internalization? Studies of KIR6.2/SUR1/2A-based KATP channels have also demonstrated PKC-dependent inhibition of KATP 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 KIR6.2 was essential for PKC-induced internalization but that PKC-mediated phosphorylation of KIR6.2 was not involved in the process. These data suggest that PKC-dependent phosphorylation of some other target participates in channel endocytosis. Recent studies of KIR6.1/SUR2B channels expressed in HEK cells indicate that this may not be the case for VSMC KATP channels.9 In this system, phosphorylation of several serine residues in the C terminus of KIR6.1 appeared essential for PKC-mediated inhibition of KATP channel currents. However, it is worth noting that channel internalization was not examined in this study and should be investigated in the future. Thus, the molecular details of how activation of PKC-ε leads to internalization of VSMC KATP channels remains to be established.
Second, what is the fate of internalized VSMC KATP channels? PKC-dependent internalization of KIR6.2/SUR1/2A-based KATP channels leads to the 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 KIR6.1/SUR2B channels expressed in VSMCs will require further investigation.
Third, is channel internalization the only means by which activated PKC inhibits KATP channels? Studies of native VSMC KATP channels3 have shown that exogenous PKC reversibly inhibits KATP channel activity by increasing the interburst interval. Because all of the 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 on washout of the PKC. Recent studies of Kv1.5 channel recycling in atrial myocytes demonstrate recovery of internalized channels with a half-time for recovery on the order of 29 minutes.10 This is considerably slower than the 5 minutes cited for recovery of VSMC KATP 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 KATP channels, perhaps independently. Additional studies will be required to resolve this issue.
Finally, as noted above, vasoconstrictors, such as angiotensin II, can also inhibit KATP channels by ≥2 additional mechanisms: receptor-mediated inhibition of constitutive adenylate cyclase activity5 and Ca2+-dependent activation of calcineurin (protein phosphatase 2B).4 Whether these pathways also involve modulation of KATP channel trafficking remains to be established.
Thus, whereas the studies of Jiao et al7 move our understanding of the mechanism by which PKC inhibits VSMC KATP channels forward, additional studies will be required to define the molecular details of PKC-induced KATP channel internalization and, critically, the importance of this process in the maintenance of cardiovascular homeostasis in health and disease.
Sources of Funding
This work was supported by Public Health Service grants HL 32469 and HL 086483.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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Wilson AJ, Jabr RI, Clapp LH. Calcium modulation of vascular smooth muscle ATP-sensitive K(+) channels: role of protein phosphatase-2B. Circ Res. 2000; 87: 1019–1025.
Sampson LJ, Davies LM, Barrett-Jolley R, Standen NB, Dart C. Angiotensin II-activated protein kinase C targets caveolae to inhibit aortic ATP-sensitive potassium channels. Cardiovasc Res. 2007; 76: 61–70.
Jiao J, Garg V, Yang B, Elton TS, Hu K. Protein kinase C-ε induces caveolin-dependent internalization of vascular adenosine 5′-triphosphate–sensitive K+ channels. Hypertension. 2008; 52: 499–506.
Shi Y, Cui N, Shi W, Jiang C. A short motif in Kir6.1 consisting of four phosphorylation repeats underlies the vascular KATP channel inhibition by protein kinase C. J Biol Chem. 2008; 283: 2488–2494.
McEwen DP, Schumacher SM, Li Q, Benson MD, Iniguez-Lluhi JA, Van Genderen KM, Martens JR. Rab-GTPase-dependent endocytic recycling of Kv1.5 in atrial myocytes. J Biol Chem. 2007; 282: 29612–29620.