Subcellular trafficking and endocytic recycling of K_ATP channels

Abstract

Sarcolemmal/plasmalemmal ATP-sensitive K⁺ (K_ATP) channels have key roles in many cell types and tissues. Hundreds of studies have described how the K_ATP channel activity and ATP sensitivity can be regulated by changes in the cellular metabolic state, by receptor signaling pathways and by pharmacological interventions. These alterations in channel activity directly translate to alterations in cell or tissue function, that can range from modulating secretory responses, such as insulin release from pancreatic β-cells or neurotransmitters from neurons, to modulating contractile behavior of smooth muscle or cardiac cells to elicit alterations in blood flow or cardiac contractility. It is increasingly becoming apparent, however, that K_ATP channels are regulated beyond changes in their activity. Recent studies have highlighted that K_ATP channel surface expression is a tightly regulated process with similar implications in health and disease. The surface expression of K_ATP channels is finely balanced by several trafficking steps including synthesis, assembly, anterograde trafficking, membrane anchoring, endocytosis, endocytic recycling, and degradation. This review aims to summarize the physiological and pathophysiological implications of K_ATP channel trafficking and mechanisms that regulate K_ATP channel trafficking. A better understanding of this topic has potential to identify new approaches to develop therapeutically useful drugs to treat K_ATP channel-related diseases.

ATP-SENSITIVE K⁺ CHANNELS

Sarcolemmal/plasmalemmal ATP-sensitive K⁺ (K_ATP) channel opening is regulated by alterations in cytosolic ATP and ADP concentrations, which couples cellular energy metabolism to membrane excitability. This property of “metabolo-electrical” coupling is essential in several biological processes, for example in pancreatic β-cells where K_ATP channels close in response to increased plasma glucose and intracellular ATP production to trigger insulin secretion (1), in glucose sensitive neurons in the hypothalamus where K_ATP channels link low glucose levels to feeding behavior (2), in the heart where K_ATP channel activation during rapid heart rates promote adaptive shortening of the cardiac action potential (3), and during metabolic impairment where K_ATP channels in the vascular smooth muscle and the endothelium contribute to vasodilatation and increased blood flow (3). In the heart, the actions of K_ATP channels are most pronounced during pathological states that involve deficits in energy metabolism. During hypoxia, for example, K_ATP channel opening rapidly shortens the cardiac action potential (3). Sarcolemmal K_ATP channels are also instrumental in mediating the cardioprotective response of ischemic preconditioning (4).

A K_ATP channel is composed of four pore-forming inward rectifier potassium channel (Kir6) subunits and four regulatory sulfonylurea receptor (SUR) subunits, which belong to the ATP-binding cassette (ABC) superfamily (Fig. 1A). The Kir6 subfamily has two members, Kir6.1 and Kir6.2, whereas the SUR subfamily has two; namely SUR1 and SUR2, with SUR2 existing as one of at least two splice variants, SUR2A and SUR2B. Pancreatic β-cells predominantly express Kir6.2 and SUR1, smooth muscle cells highly express Kir6.1 and SUR2B, and cardiomyocytes express all subunit isoforms but are relatively abundant in Kir6.2 and SUR2A (3).

Figure 1.

Topology and structural information of Kir6.x and SURx channels. A: topology of the SURx/Kir6.x channel with domains labeled and colored to match the three-dimensional (3-D) structures shown below. The branched sticks represent the location of the two N-linked glycosylation sites in SUR. The sky-blue letters represent the signaling motifs involved in ATP-sensitive K⁺ (K_ATP) channel trafficking. The brown dashed line represents the interacting domains between SUR and Kir6 in the assembled K_ATP hetero-octameric complex. Also shown are some of the trafficking motifs identified in the K_ATP channel subunits that regulate K_ATP channel anterograde trafficking, membrane anchoring, and endocytosis. The motif sequences shown are referred to human Kir6.2 and SUR1. B: cryoEM structure of the K_ATP channel based on a structural model of the pancreatic K_ATP channel generated using PDB ID 6BAA, with the core Kir6.2 tetramer forming the potassium ion pore (gray). The outer regulatory proteins SUR1 has three transmembrane domains, TMD0 (forest green), TMD1 and TMD2 (pale green). Each Kir6.2 subunit has two transmembrane helices, the inner helix (M2) and outer helix (M1), an intermembrane pore helix. ATP is bound to Kir6.2 (yellow spheres), and the K⁺ ion path is closed. Glibenclamide (GBC) is captured bound to the channel (purple spheres). ER, endoplasmic reticulum.

SUBCELLULAR TRAFFICKING OF MEMBRANE PROTEINS

The total amount of current through a particular channel type is determined by

$$I=n·i·P_{\text{o}},$$ (1)

where I represents the amount of ionic current in a cell, n the number of available channels, i the unitary channel current, and P_o the average open probability of each channel.

Many studies aim to understand how K_ATP channels are regulated by alterations in P_o (e.g., during disease states, with pharmacological interventions, with receptor signaling, etc.). It is becoming increasingly clear that the number of channels (n) is not fixed and can be dynamically altered by de novo protein synthesis, degradation of existing channel subunits, and by endocytic recycling of channels from intracellular compartments to and from the surface. Cells internalize the equivalent of their entire surface area up to five times per hour and most of the endocytosed proteins are recycled back to the membrane (5, 6). Not only is endocytic recycling a rapid process occurring at a time scale of minutes, but the rates of endocytosis and exocytosis are tightly controlled by specialized trafficking proteins and signaling pathways (6). Thus, endocytic recycling is a potentially powerful mechanism to regulate the surface density and availability of ion channels (7). In the case of the K_ATP channel, the major regulators of function were long thought to directly affect P_o, for example, block by intracellular ATP, activation by intracellular ADP, activation by PIP₂, opening by K_ATP channel openers such as diazoxide or pinacidil, and block of the channels by sulfonylureas such as tolbutamide or glibenclamide (3). It is becoming increasingly apparent, however, that some of these factors also affect endocytic recycling and surface expression. Moreover, sulfonylureas not only decrease P_o but also enhance channel biogenesis and assembly efficiency thus increasing surface expression, whereas channel openers tend to decrease channel surface expression. This review extends previous publications (3, 8) by highlighting studies demonstrating that modulating the number of available channels (n) is a major regulator of the K_ATP channel current under both physiological and pathophysiological conditions.

PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL RELEVANCE OF K_ATP CHANNEL TRAFFICKING

There are several studies demonstrating that K_ATP channel surface density is dynamically regulated during health, and can be negatively impacted by disease.

Glucose-Induced Trafficking in the Pancreatic β-Cell

The role that K_ATP channels have as a regulator of insulin secretion from pancreatic β-cells in the islet of Langerhans is well established. During fasting states, when intracellular ATP levels are low, the K_ATP channel is open, which clamps the membrane potential to negative values, and in turn mitigates electrical activity of the β-cell. After a meal, glucose uptake and alterations in the ATP and ADP levels inside the cell causes the K_ATP channel open probability to decrease, which depolarizes the β-cell and sets in motion a cascade of events that includes activation of other ion channels and transporters, action potential firing, increases in intracellular Ca²⁺, and vesicular release of insulin from intracellular granules (9). Hormones released from the gut after a meal, such as glucagon-like peptide 1 (GLP-1), sensitize this process, in part by further decreasing the K_ATP channel open probability through a noncanonical cAMP/EPAC-mediated pathway (10). What is not commonly appreciated, however, is that changes in K_ATP channel open probability are mirrored by corresponding trafficking alterations and that the change in the number of surface channels (n) may be as important, if not more important, than the changes in activity (Fig. 2). In one of the earliest studies demonstrating a link between glucose and trafficking, glucose stimulation was found to increase K_ATP surface expression (11). A more recent study, however, suggests the opposite namely that the surface expression of Kir6.2 is profoundly increased by glucose deprivation, an intervention that also decreases the open probability of the channel, without changes in the total Kir6.2 protein level (12). This process is reversible and occurs with a relatively rapid time scale (in the minute range), strongly indicative that trafficking mechanisms are involved. Intracellular energy homeostatic changes caused by glucose deprivation lead to an increase in intracellular AMP levels, which activates AMP-activated protein kinase A (AMPK) (13). This pathway turns out to be key: apart from the fact that AMPK strongly enhances the open probability of K_ATP channels (14), AMPK activation also plays an integral role in increasing the surface expression of K_ATP channels upon glucose deprivation in pancreatic β-cells (12). This action of AMPK goes beyond glucose deprivation. Leptin, a hormone released by fat cells and is known to regulate insulin secretion, also increases the K_ATP channel activity (15). Consistent with a trafficking mechanism, an early study has demonstrated that leptin activation of K_ATP channels involves disruption of the actin cytoskeleton (16). Indeed, subsequent studies have shown that leptin transiently increases the K_ATP channel surface expression in a manner that depends on AMPK activation (17, 18) and Rac-mediated remodeling of the actin cytoskeleton (19). This increase results primarily from increased channel trafficking to the plasma membrane rather than reduced endocytosis of surface channels. Thus, surface trafficking is a necessary element to regulate K_ATP channels in pancreatic β-cells.

Figure 2.

Signaling pathways involved in ATP-sensitive K⁺ (K_ATP) channel trafficking in the pancreatic β-cell. In the pancreatic β-cell, AMP-activated protein kinase A (AMPK) is activated by phosphorylation at T172 site either in low glucose conditions where AMP levels are elevated, or in the presence of adipocyte hormone, leptin (at 6–11 mM glucose). Leptin binding to its biospecific receptor elicits a downstream signaling cascade that involves an increase in Ca²⁺ influx into the cells through either activation of surface TRPC4 channels or potentiation of NMDA receptors (NMDAR) activity, which activates CaMKKβ leading to phosphorylation and activation of AMPK. Active AMPK elicits activation of AKAP79/150-plasma membrane-anchored PKA, which then triggers depolymerization of the actin cytoskeleton to allow vesicles containing K_ATP channels to traffic to the plasma membrane.

Subcellular Trafficking in Vascular Smooth Muscle

Vascular smooth muscle expresses K_ATP channels that consist of a combination of Kir6.1 and SUR2B subunits (20). There is ample evidence that K_ATP channels regulate smooth muscle tone and blood flow, particularly in small vessels. During hypoxia, for example, increased K_ATP open probability causes smooth muscle relaxation (21). PKA signaling, which causes coronary vasodilation, also stimulates vascular K_ATP channel open probability (22). By contrast, PKC signaling, associated with vasoconstriction, inhibits vascular K_ATP channel opening. In parallel with the decreased open probability of vascular K_ATP channels, PKC also decreases the number of surface K_ATP channels (23). In the latter study, PKC activation with phorbol esters led to a decrease in K_ATP channel current, which was prevented by a dominant-negative dynamin mutant. Internalization was also observed in response to the vasoconstrictor, angiotensin II, demonstrating a role for internalization by endocytosis as a key regulator of vascular K_ATP channel function. Apart from this single study, little is known about regulated surface expression of vascular K_ATP channels.

Subcellular Trafficking in Cardiomyocytes

Spatially defined subcellular expression patterns.

Cellular functions often depend on the distribution of ion channels, transporters, and pumps to specialized areas of the cell membrane. For example, voltage-gated Na⁺ and K⁺ channels are concentrated at the nodes of Ranvier, which are essential for saltatory conduction in myelinated nerves, whereas nicotinic acetylcholine receptor channels are mainly at the postsynaptic membrane of the neuromuscular junction, to initiate excitation-contraction (EC) coupling of skeletal muscle. Targeted trafficking of several membrane proteins occurs in cardiac myocytes and the currently proposed mechanisms are summarized in Fig. 3. For example, there is a distinct subpopulation of L-type Ca²⁺ channels (LTCCs) present at the transverse (T)-tubule, where their close proximity with ryanodine receptors (RYR2) of the sarcoplasmic reticulum (SR) allows for Ca²⁺-induced Ca²⁺ release from the SR and cardiac contractions (24). Some LTCCs, however, are targeted to another physically and functionally distinct location, namely to lipid rafts and caveolae (25), where they are subject to local regulation by signaling pathways (26, 27). Separate pools of Na⁺ channels have also been identified in cardiac myocytes (28). One pool is targeted to the lateral membrane by the syntrophin/dystrophin complex (29, 30). Another subpopulation of Na⁺ channels is organized in a highly specialized macromolecular complex at the intercalated disk (ICD) (31), where targeting and anchoring is coordinated by AnkG, SAP97, EB1, and PKP2 (28, 32). The concept of regional specification of channels has recently been extended to K⁺ channels. For example, some inward rectifier K⁺ channels (Kir2.1) localize to the perinexus (33) and may co-traffic with Nav1.5 (34). A subpopulation of another class of K⁺ channels (the Kv2 delayed rectifier) clusters in specific membrane subcompartments in cardiomyocytes (35) and neurons (36), whereas other Kv2 channels locate to lipid rafts (37). There is now evidence that the clustered Kv2 channels are in fact nonfunctional (38) and serve an important scaffolding role, interacting with VAP proteins (VAMP-associated proteins) to initiate membrane/endoplasmic reticulum (ER) contact sites (39, 40), which have essential roles in a variety of biological processes (41). Thus, understanding the channel composition and their presence at specific membrane compartments within a cardiac myocyte provides new insights into the mechanism of electrical excitation and may pave the way for novel therapies against cardiac rhythm disturbances. There may also be distinct K_ATP channel subpopulations within a cardiac myocyte. Similar to the cardiac Na⁺ channel, K_ATP channels localize to at least two subcellular membrane domains in a ventricular myocyte (Fig. 3).

Figure 3.

Targeted trafficking of channels in cardiac myocytes. Depicted is a cardiac myocyte that connect to its immediate neighbors in an end-to-end manner at the intercalated disk (ICD). De novo synthesized membrane proteins can be targeted to specific subcellular regions by anterograde trafficking mechanisms and by anchoring to specific subcellular domains. Shown are targeted delivery via myosin or microtubule based mechanisms and targeted delivery to lateral membranes, the specialized t-tubule structures or the ICD. Channels can be stabilized at specific regions through anchoring mechanisms [e.g., binding to ankyrin-B (Ank-B) or ankyrin-G (Ank-G)] or by localization to specific subcellular domains, such as caveolae. Our understanding of ATP-sensitive K⁺ (K_ATP) channel subcellular trafficking in a cardiac myocyte is still in its infancy.

The presence of K_ATP channels in lateral membranes was established by their initial identification with patch clamp methods (42). Early scanning ion conductance microscopy, combined with whole cell voltage clamping, suggested that lateral K_ATP channels are present as submicrometer clusters in Z-grooves of the sarcolemma (43). Moreover, the Kir6.2 subunit of K_ATP channels was shown to physically interact with ankyrin-B (AnkB) (44, 45), which is expressed at lateral membranes in cardiac myocytes and localizes to Z- and M-lines in an isoform-dependent manner (46). Finally, some K_ATP channels localize to caveolae (47, 48), which are present in t-tubules and lateral membranes (49).

K_ATP channels are also present at the ICD region (Fig. 3), which links cardiac myocytes end-to-end, ensures rapid propagation of electrical impulses between cells, and allows mechanical coupling sufficiently strong to withstand the mechanical forces during cardiac contraction. While searching for novel K_ATP channel interactors with mass spectrometry approaches, we previously identified desmosomal proteins, including plakoglobin (PG) and plakophilin-2 (PKP2) (50, 51). We verified that they physically interact with K_ATP channels with coimmunoprecipitation assays (50, 51). With differential centrifugation assays under detergent-free conditions, we found that rat ventricular K_ATP channel subunits cosegregate with PKP2, but not with markers of other ICD substructures including Cx43 (gap junctions). Kir6.2 colocalizes with PG and PKP2 in confocal immunofluorescence microscopy. Using super-resolution microscopy techniques, we demonstrated that K_ATP channels cluster within nanometer distances from these junctional proteins at the ICD region of cardiomyocytes (51). From recordings made in the inside-out or in the cell-attached patch configuration, we found that the local K_ATP channel density in membrane patches at the ICD region of isolated mouse or rat cardiomyocytes is substantially higher than at lateral membranes, and that this difference is mitigated in PKP2^+/− mice (51).

Functional interaction between K_ATP channels and Na⁺ channels at the ICD.

A theme in channel biology that has received much recent activity is that ion channels, exchangers, and pumps present in the same subdomain have the potential to physically and functionally interact with each other. For example, there are numerous studies demonstrating functional interaction between K_ATP channels and the Na⁺/K⁺ pump in a variety of cell type, including cardiac myocytes (52–58). Although physical interaction between these proteins is suggested by mass spectrometry data showing that the Na⁺/K⁺ ATPase α-subunit was present in K_ATP channel immunoprecipitates (50), the structural basis and mechanisms of physical interaction between these two proteins remain to be elucidated. The enrichment of both Na⁺ and K_ATP channels at the cardiac ICD and the fact that both interact with desmosomal proteins and are similarly affected by PKP2 deficiency (51, 59, 60) suggested the possibility that these two channels may functionally interact. Indeed, these two channels were found to reciprocally regulate each other in heterologous expression systems. Overexpressing the K_ATP channel subunits led to a decrease of Nav1.5 Na⁺ channel subunit, and overexpression of Nav1.5 led to a decrease of Kir6.2/SUR2A channel current. The relevance of this finding to cardiomyocytes was demonstrated by the finding that overexpression of Kir6.2 by adenoviral delivery significantly reduced the whole cell Na⁺ current density in cardiomyocytes (61). Using super-resolution microscopy techniques, it was found that native Na⁺ and K_ATP channels co-cluster at the ICD of cardiac myocytes. Mechanisms of anchoring and co-clustering will be discussed in Anchoring Mechanisms. Although the functional relevance of co-clustering of these two channels is still under investigation, this study provides a paradigm in which to consider the pharmacological and genetic aspects of arrhythmogenesis.

Dynamic changes in surface expression during physiological conditions.

Changes in surface expression of K_ATP channels in cardiac myocytes under physiological conditions are suggested from the literature, but not well studied. For example, exercise training of mice for only 5 days leads to an approximately two times increase in the number of K_ATP channels per patch in isolated cardiac myocytes, with relatively small changes in mRNA expression (62). Given that AMPK is activated with exercise training, and that the positive regulation of K_ATP channel surface expression can occur via this pathway (see the previous section), the untested possibility is raised that elevated surface expression occurred. Regardless of the mechanism, the physiological consequence of the increased surface expression of K_ATP channels with exercise is clear. With an elevated heart rate, such as during strenuous exercise, action potential duration adaption occurs over the time course of a few minutes (63). This progressive action potential shortening allows for adequate filling time for the ventricle, and is responsible for a decreased refractoriness and prevention of arrhythmias. Loss of K_ATP channel activity with glibenclamide, or cardiac-specific overexpression of dominant-negative Kir6 subunits strongly mitigates action potential frequency adaptation (62). There is a strong possibility that AMPK-mediated changes in K_ATP surface expression underlies this response and future studies are needed to investigate this possibility and the potential role(s) of altered K_ATP channel surface expression in other physiological processes.

Dynamic changes in surface expression during pathophysiological conditions.

Dynamic alterations in K_ATP channel surface expression in cardiomyocytes under pathophysiological conditions is an emerging topic in the literature (Fig. 4). The role of K_ATP channels in the cardioprotective effects of ischemic preconditioning (IPC) has been well established—both using pharmacological and genetic approaches (3). To our knowledge, one of the first studies demonstrating that an increase in surface K_ATP channels underlies the protective effects of IPC came from a study using a mouse model with transgenic overexpression of a specific CaMKII inhibitory peptide AC3-I (64). Cardiomyocytes from the transgenic mice exhibited significantly increased I_K,ATP current density and greater sarcolemmal expression of Kir6.2, without significant changes in total Kir6.2 expression or alterations in open probability or other channel properties. The AC3-I transgenic hearts were protected against infarct development after ischemia/reperfusion to the same extent as with a K_ATP channel opener. Although direct evidence was not provided, this study strongly suggested that myocardial CaMKII inhibition confers resistance to ischemia-reperfusion injury (similar to IPC) by enhancing the surface expression of K_ATP channels (64). Acute upregulation of I_K,ATP density was directly demonstrated in a cellular model of pharmacological preconditioning using isolated rat cardiac myocytes (65). In the latter study, pretreatment of myocytes with an α1 adrenergic receptor agonist phenylephrine afforded improved protection against cell death after oil-overlay ischemic pelleting, which was prevented by a K_ATP channel blocker, strongly suggesting that the enhanced K_ATP channel surface expression underlies protective effects of preconditioning. Interestingly, a role for AMPK, PKC-δ, and p38 was suggested to underlie the increase in I_K,ATP current induced by phenylephrine preconditioning (65).

Figure 4.

Mechanisms that regulate ATP-sensitive K⁺ (K_ATP) channel subcellular trafficking in a cardiac myocyte during ischemia and ischemic preconditioning. Prevailing data show that sarcolemmal K_ATP channels are protective against metabolic stress, possibly by reducing Ca²⁺ overload (e.g., during ischemia) and/or preserving mitochondrial function. Ischemia appears to mitigate the protective effects of K_ATP channels by causing internalization through endocytosis, mediated by a CaMKII-dependent mechanism. One of the cardioprotective mechanisms of ischemic preconditioning may reside in the activation of a PKC/AMPK/p38 signaling axis, which promotes K_ATP channel surface expression, which counteracts the detrimental ischemia-induced internalization of K_ATP channels from the sarcolemma. Further research into this proposed mechanism is needed, which could lead to new therapeutic approaches to develop cardioprotective strategies.

An interesting twist to the tale came about when the mechanism by which CaMKII regulates K_ATP channel surface expression was elucidated. The Zingman’s laboratories demonstrated that the β1 adrenergic receptor agonist isoproterenol led to a reversible inhibition of whole cell I_K,ATP in isolated mouse ventricular cardiomyocytes over a time course of minutes and that this inhibition did not occur in myocytes overexpressing the CaMKII inhibitory peptide AC3-I (66). An acute trafficking mechanism was suggested by the finding that surface density of Kir6.2 and SUR2A (measured with surface biotinylation assays) was decreased by CaMKII activation. Indeed, a role for the Ca²⁺/CaMKII pathway in stimulating endocytosis was demonstrated by the finding that internalization was prevented by a dominant negative dynamin peptide or the endocytosis inhibitor, dynasore. A tyrosine-based motif was identified as mediating endocytic internalization (66) (see Sorting and Anchoring Motifs in K_ATP Channel Subunits).

A study with cardiac-specific Kir6.2 knockout mice clarified the role of K_ATP channels in the perspective of cardiac ischemia and IPC (4). As expected, hearts from wild-type mice were protected from infarct development after ischemia by IPC perfusion protocols or by adenosine (67). Cardiomyocyte-specific Kir6.2 knockout did not mitigate infarct development after ischemia, but reversed the protective effects of IPC or adenosine, demonstrating a key role for K_ATP channels in the protective mechanisms. In parallel, K_ATP channel subunit expression was measured in membrane fractions obtained with density gradient centrifugation techniques. K_ATP channel subunits were present both in fractions enriched with sarcolemmal proteins and in membrane fractions that contain endosomal proteins. In an early study, surface K_ATP channel expression was found to be increased during ischemia (68). Experimental conditions were largely uncontrolled in the latter study, however, since the ischemia model used was to excise hearts from rats 20–30 min postmortem. When performing experiments under laboratory controlled conditions (temperature, ionic compositions, etc.), it was found that ischemia actually reduces the K_ATP channel content in sarcolemmal fractions, which was prevented by a cocktail of trafficking inhibitors (brefeldin A, colchicine, and nocodazole) or by the inhibitor of endocytosis, dynasore. Moreover, consistent with the findings discussed in the previous paragraph, CaMKII inhibition also prevented ischemia-induced K_ATP channel internalization. Thus, unlike the paradigm in pancreatic β-cells and smooth muscle cells, where K_ATP channel activity parallels surface expression changes (i.e., reduced channel activity is associated with internalization), the reverse seems to occur in cardiomyocytes, where the expected activation of K_ATP channel opening during ischemia (69) is associated with internalization. This is a counterintuitive phenomenon since K_ATP channels are cardioprotective (70) and internalization would mitigate this protective mechanism. The same study highlighted a mechanism by which K_ATP channels contribute to the protective effects of ischemic preconditioning. Building on the idea that endosomal K_ATP channels may serve as a reservoir for recycling of channels to the surface (68), a key finding was made that the internalized K_ATP channels are restored to the cell surface with ischemic preconditioning or by adenosine pretreatment (4). This process was PKC dependent, highlighting at least one of the mechanisms by which PKC activation participate in the protective effects of preconditioning. Overall, these studies make a strong case for a role of subcellular trafficking and endocytic recycling in the well-described protective effects of K_ATP channels.

The time course of dynamic regulation of K_ATP channel turnover in cardiac myocytes has not been studied formally, mainly due to the challenges of studying native channels with currently available antibodies. Experiments performed with heterologous expression systems suggest a rapid turnover rate in the order of minutes (see mechanisms of subcellular trafficking and endocytic recycling of k_ATP channels). Experiments with an electrophysiological approach in cardiomyocytes showed that CaMKII-dependent internalization (and reversal) similarly occurs in a time scale of minutes (66), suggesting that dynamic regulation of K_ATP channel surface expression is rapid in the heart. Experiments with antibody uptake and recycling or similar approaches are needed to verify this expectation. Such experiments are expected to be challenging given the compact nature of the cardiomyocyte microarchitecture and the limitations of the available antibodies against native K_ATP channels.

Trafficking Defects Caused by Genetic Variants in K_ATP Channel Genes

Variants in K_ATP channel genes, KCNJ8, KCNJ11, ABCC8, and ABCC9 are associated with several disease states and syndromes in humans (3, 71). The most extensively studied are those in KCNJ11 and ABCC8 encoding Kir6.2 and SUR1, respectively, which form the predominant Kir6.2/SUR1 K_ATP channel subtype in pancreatic endocrine islets and neurons. Variants that cause loss of channel function (LOF) underlie congenital hyperinsulinism, which is characterized by persistent and unregulated insulin secretion and life-threatening hypoglycemia (72). By contrast, variants resulting in gain of channel function (GOF) are the most common cause of neonatal diabetes mellitus (73), found in ∼40% of patients diagnosed with permanent diabetes at <6 mo of age (74). Some GOF mutations also lead to developmental delay, epilepsy in additional to neonatal diabetes, known as the DEND syndrome (74). Although many of the loss- or gain-of-function variants affect K_ATP channel gating, many are found to prevent or reduce channel trafficking to the plasma membrane (75) especially those associated with congenital hyperinsulinism. Counterintuitively, several neonatal diabetes/DEND syndrome associated mutations also reduce channel expression at the plasma membrane. With these mutations, the severe gating defects override reduced surface expression to yield an overall gain of function disease phenotype (76–78). For most trafficking mutations reported to date, misfolding or misassembly of channel subunits leading to ER retention and proteasome degradation of mutant proteins account for the trafficking defect. However, a Kir6.2 mutation E282K at a di-acidic ER exit signal, ²⁸⁰DLE²⁸² has been reported to disrupts Sar1-GTPase-dependent ER exit (79). Of note, trafficking defects of a subgroup of ER-retained mutants can be corrected by treatment with antidiabetic sulfonylureas or other pharmacological chaperone molecules (75). These mutations are seen clustered at the SUR1-TMD0 and Kir6.2 TM1 helix where the two subunits make contact in recent cryoEM structures of the channel (Fig. 1B) (80, 81). Mutations at this interface likely interfere with channel assembly. In cryoEM structures of channels bound to pharmacological chaperones, the distal N-terminus of Kir6.2 lies in a cleft formed by the two transmembrane domains of the ABC core of SUR1 and adjacent to the chaperone bound in the transmembrane helix bundle above the first nucleotide binding domain. The distal N-terminus of Kir6.2 has been shown to be involved in K_ATP channel assembly (82, 83) and high affinity binding of sulfonylureas (84). Thus, sulfonylureas and other K_ATP pharmacological chaperones likely facilitate assembly of mutant channels by stabilizing interactions between Kir6.2-N terminus and the SUR1 ABC core to correct mutant channel trafficking defects (81).

Additional diseases, including Brugada syndrome, ventricular fibrillation with early repolarization, early infantile epileptic encephalopathy, sudden infant death syndrome, heart failure, dilated cardiomyopathy, familial atrial fibrillation, coronary spasm, acute myocardial infarction (MI), Cantú syndrome, and AIMS (ABCC9-related Intellectual disability Myopathy) syndrome have been linked to variations in K_ATP channel genes (3, 71). Among these, Cantú syndrome has been most definitively linked to KCNJ8 and ABCC9 mutations causing gain of function of vascular K_ATP channels comprising Kir6.1 and SUR2B (85). Whether any of the genetic variants associated with the aforementioned diseases affect trafficking and surface expression of the resulting channels remain largely unexplored.

MECHANISMS OF SUBCELLULAR TRAFFICKING AND ENDOCYTIC RECYCLING OF K_ATP CHANNELS

The surface expression of K_ATP channels is regulated by a fine balance between synthesis, assembly, anterograde trafficking, membrane anchoring, endocytosis, endocytic recycling, and degradation (Fig. 5). There are multiple trafficking steps and each of these is tightly controlled by enzymatic activity, subunit interaction, specific amino acid targeting motifs present in the transported cargo, posttranslational modifications of trafficking proteins, and amino acid mutations causing misfolding defects of the cargo. In the case of the K_ATP channel, an additional element to consider is that there are multiple possible subunit combinations of Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B and that isoform-specific sequence differences and assembly may result in shared and cell-specific trafficking mechanisms. Factors that determine surface expression density of membrane proteins include the rate of protein synthesis, modifications taking place within the endoplasmic reticulum (ER), and the activity of trafficking proteins that interact with specific membrane-bound cargos. These determine the rate of the anterograde trafficking pathway (when a protein travels from the ER to the plasma membrane through the Golgi apparatus). Protein stability at the plasma membrane is determined in part by anchoring mechanisms and/or localization in specific subcellular domains (e.g., t-tubules, the ICD, or caveolae). The presence of channels or transporters at the membrane is a transient phenomenon since all membrane proteins are endocytosed, after which they can be recycled to the plasmalemma or trafficked to the cell’s protein degradation machinery.

Figure 5.

A simplified depiction of some of the subcellular processes responsible for ATP-sensitive K⁺ (K_ATP) channel endocytic recycling. De novo synthesized K_ATP channel complexes are assembled in the endoplasmic reticulum and are directed to the plasma membrane via the Golgi apparatus, where specific posttranslational modifications (e.g., glycosylation) take place. Surface channels can be stabilized by several anchoring mechanisms (not shown here). Multiple lines of evidence suggest that plasmalemmal K_ATP channels are internalized through a dynamin- and clathrin-dependent endocytic process. Endocytosed vesicles are likely directed to early endosomes, from where trafficking can be directed to late endosomes (which may serve as a subcellular buffer) or to the endocytic recycling compartment, from where channels may be redirected for surface expression. There are multiple trafficking steps, proteins controlling these steps, and their regulation by receptor signaling pathways that remain to be elucidated.

Transcriptional Regulation, Protein Stability, and Degradation

Regulation of K_ATP channels by transcriptional alterations are well documented, but not well studied mechanistically. Prevailing data show that transcription of Kir6.2 mRNA can be upregulated by mechanical stress (86). The subunit expression of K_ATP channels is also altered following hypoxia or ischemia. In H9c2 rat heart cells, hypoxia upregulates mRNA expression of Kir6.1 and SUR2A (87, 88). These changes occur with a relatively slow time course. For example, in a model of left coronary occlusion of rat hearts upregulation of Kir6.1, SUR1, and SUR2A mRNA was reported to occur after 8–20 wk postinfarction (89). Similarly, a strong increase in Kir6.1 mRNA expression was observed 2–6 h after renal ischemia in the rat (90), and in rat heart, Kir6.1 mRNA expression was upregulated after 60 min of myocardial regional ischemia followed by 24–72 h (but not 3–6 h) of reperfusion (91). The latter study reported that Kir6.2 and SUR2 mRNA expression levels were unchanged. The local renin-angiotensin system was described to have a role in this process (92). Transcription factors that have been implicated in K_ATP channel mRNA expression include forkhead box (FOX) O1 and hypoxia-inducible factor (HIF)-1α (93, 94).

Protein stability may also influence K_ATP channel availability. This is not a rate-limiting factor, however, since the half-life of Kir6.2/SUR1 and Kir6.2/SUR2A channels is ∼15–20 h (68, 95–97). When expressed by itself, the half-life of Kir6.2 is ∼2 h, suggesting that association with SURx stabilizes the protein complex (98). Kir6.1 protein levels are upregulated in hearts and brains of rats subjected to chronic hypoxia, whereas Kir6.2 protein expression levels are downregulated by hypoxia (89). It has been suggested that tissue hypoxia may operate through posttranscriptional mechanisms by inhibiting Kir6.2 protein degradation (86). The mechanisms of K_ATP channel expression can be twofold by involving both lysosomal pathway as has been shown upon PKC activation (99) and the ubiquitin-proteasome degradation pathway since degradation of both Kir6.2 and SUR1 is mitigated by the proteasome inhibitor MG132 (96, 98).

The expression and function of K_ATP channels in the hours to days following ischemia/reperfusion is an important topic for future studies. Importantly, patients experiencing a myocardial infarction are at an increased risk for a second cardiac event in the post-MI period (e.g., re-infarction, heart failure, and sudden death). This occurs despite the availability and use of effective methods for limiting infarct size with thrombolytic agents and primary angioplasty. Given the protective role of K_ATP channels in stress events, it is essential to understand how their expression and function is altered following an ischemic episode. It is clear, however, that these transcriptional and posttranscriptional changes are slow relative to the dynamic alterations in endosomal recycling, which represents another important therapeutic target.

Folding and Assembly in the Endoplasmic Reticulum

Proper folding, essential posttranslational modifications, and correct assembly of multisubunit complexes are necessary steps for membrane proteins to exit the endoplasmic reticulum and to be forwarded along the anterograde trafficking pathway to the plasmalemma. Soon after the molecular identification of K_ATP channel subunits it was realized that Kir6.x and SURx subunits need to be co-expressed to form functional channels (100). Mechanistic insight was provided by the finding that co-assembly of Kir6/SUR subunits is needed for effective membrane trafficking (101). The latter study has identified a specific ER retention motif (RKR) in both Kir6.x and SURx subunits (RXR in SUR2). These motifs were proposed to hide in the co-assembled protein, thus allowing the protein complex to escape the ER and enter the anterograde pathway. Unfortunately, these motifs are not well resovled in the cryoEM structures reported to date to confirm this notion. Aside from this ER retention motif, a di-acidic motif (DXE) in Kir6.2 was found to function as an ER exit signal, which concentrates K_ATP channels into COPII-enriched ER exit sites before ER export via a process that requires Sar1-GTPase (79). Some reports suggested that SUR subunits may also have an ER exit motif (LLXXXXXXF) that functions as an anterograde signal, required for K_ATP channels to exit the ER/Golgi compartments (102). This result is controversial, however, since the distal C-terminus of SUR1 was reported not to be required for efficient surface expression (82). A pair of conserved cysteine residues at the N-terminus of SUR1 (C6 and C26), initially identified in ABCB6, forms an intramolecular disulfide bond critical for ER processing (103). Indeed, the C6G mutation in patients shows greatly reduced surface K_ATP channel expression (8). The interesting possibility was raised that these and other trafficking motifs might serve to create a reservoir of intracellular channels to regulate and buffer the number of available surface channels (101). Even though studies reviewed here are making headway to understanding this phenomenon, there remains many open questions and there is much work to be done to gain a full understanding.

Amino acid substitutions due to missense variants in K_ATP channel genes may cause trafficking defects (see Trafficking Defects Caused by Genetic Variants in K_ATP Channel Genes). These mutations may cause misfolding defects leading to slow trafficking or targeting for degradation. Misfolded K_ATP channels may form complexes with Derlin-1 and an associated p97 protein, which promotes K_ATP channel degradation (104). Consistent with the role of p97 in protein ubiquitination, ubiquitin proteasome inhibitors or Derlin-1 knockdown slows the degradation rate and increases surface expression of K_ATP channels in pancreatic β-cells or heterologous expression system (96, 104).

Anterograde Trafficking

Little is known about the anterograde trafficking of K_ATP channels, but there is indirect evidence from the studies with SUR1 glycosylation. SUR1 is glycosylated at two N-linked glycosylation sites (105). The mature, complex glycosylated form of SUR1 can be distinguished from the core-glycosylated form with SDS-PAGE (101). The N-glycosylation is sensitive to Endo H digestion when occurs in the ER, but is insensitive to Endo H after modification in the Golgi. Since the mature form of SUR1 resist Endo H digestion (102, 106), one can assume that K_ATP channels traffic through the Golgi. A few regulators of anterograde trafficking have been identified. The ER-retention signal (RKR) can be recognized by both COPI vesicle coat and 14-3-3 proteins (107, 108). This competitive binding results in promoted K_ATP channel anterograde trafficking by 14-3-3 while inhibited by COPI (109). Syntaxin-1A, a SNARE protein, binds to the intracellular nucleotide binding domains of SUR1 and SUR2 (110, 111), impairs maturation and anterograde trafficking of K_ATP channels by retaining K_ATP channels in the endoplasmic reticulum-Golgi intermediate compartment (112). Finally, Rab8a was shown to be involved in membrane trafficking of Kir6.2 in the MIN6 insulinoma cell line, but the subcellular mechanism is unresolved (113). Exploring anterograde trafficking mechanisms must be a focus for future research since there are many unresolved questions. For example, we know that targeted delivery of certain proteins (e.g., Cx43 and Nav1.5) to the cardiomyocyte intercalated disk is by microtubule-based vesicle transport pathways and a microtubule plus-end binding protein EB1 (31, 114), but how K_ATP channel trafficking to this subcellular structure is determined is not yet known (Fig. 3).

Internalization

Even though K_ATP channels are anchored at the surface membrane, they are continuously being internalized and recycled to the membrane. In an antibody uptake experiment with transfected cells, ∼50% of SUR1/Kir6.2 channels was found to be internalized within 5 min, reaching a maximum by 15 min. SUR2-containing channels are internalized even more rapidly than SUR1-containing channels (68, 115). Although it was initially suggested that K_ATP channels are internalized via a caveolar-based mechanism (23), nystatin, which blocks caveolae-mediated endocytosis, does not reduce internalization (116). The reality is that caveolae are highly immobile membrane microdomains and are generally not thought to be involved in constitutive endocytic trafficking (49). The current consensus is that K_ATP channels are internalized through a dynamin- and clathrin-dependent endocytosis pathway. Readers are referred to an excellent review on this topic (117). In brief, the adaptor complex AP-2 is recruited to the inner membrane face, which serves to link clathrin to the membrane and to coordinate the structural assembly of the clathrin coat with the selection of specific cargo proteins and lipids. The membrane develops curvature until a deeply invaginated clathrin-coated pit is formed. Dynamin GTPase mediates fission of the coated pit to form the newly budded endocytotic vesicle that can be further transported in the endocytic recycling process. Evidence that K_ATP channels are internalized through this pathway include observations that 1) internalized K_ATP channels colocalize with clathrin, 2) the Kir6.2 C-terminus directly interacts with the μ2 subunit of the AP2 adaptor complex, and 3) a dominant-negative form of μ2 subunit of the AP2 adaptor complex (D176A/W421A), but not the wild-type μ2, inhibits K_ATP channel endocytosis (115). A role for dynamin is demonstrated by the findings that endocytosis can be prevented by dominant negative dynamin-1 and dynamin-2 (K44A), but not their WT versions (115), by a dominant negative dynamin peptide (66) and by the dynamin inhibitor dynasore (4, 116, 118). Other indirect evidence that K_ATP channel endocytosis occurs through a clathrin-dependent pathway includes the finding that it is blocked by chlorpromazine or potassium depletion (116). It is not clear whether SUR contains endocytosis motifs. Some studies suggested that SUR1 expressed by itself (with a mutated ER retention signal) does not internalize by itself, but can be internalized when coexpressed with Kir6.2 (115). Another study, however, suggested that SUR1 by itself with the ER retention signal mutated (RKR to AAA mutant) can undergo endocytosis independent of Kir6.2 (116). There appears to be better consensus with Kir6.2 since endocytosis of an HA-tagged Kir6.2 truncation mutant (Kir6.2Δ36) expresses better at the cell surface in the absence of SURx subunits (66). Moreover, a chimeric protein of CD4 with the Kir6.2 C-terminus (178–364; lacks RKR) can be endocytosed, whereas CD4 by itself is not (115). Internalization of Kir6.1 channels remains to be studied.

Endocytic Recycling

The majority of endocytosed membrane proteins recycle back to the surface membrane (6). Endocytosed proteins first enter early endosomes (Fig. 5). Some proteins are directly and efficiently recycled to the plasma membrane by Rab4 from the early endosomes, including internalized or “desensitized” β₂-adrenergic receptors (119, 120). Most other cargo is recycled back to the plasma membrane via a somewhat slower pathway through the endocytic recycling compartment (ERC). An antibody capture assay showed that K_ATP channels are recycled within ∼10 min (99), suggesting that K_ATP channels recycle through the ERC. Indeed, K_ATP channel subunits colocalize with Rab11 (48), which is often used as an ERC marker (121) but can also influence trans-Golgi network-to-plasma membrane transport (122). Moreover, the GFP-Rab11-S25N dominant interfering mutant, known to prevent exit of cargo from the ERC (123), led to accumulation of K_ATP channels in the ERC, strongly implicating this pathway in K_ATP channel recycling, similar to the well-described Rab11B-mediated pathway for recycling of transferrin (124). Other channels that follow a Rab11-dependent recycling pathway include Kv1.5 (125–127), KCNQ1/KCNE1 (128), hERG (129), HCN4 (130), TRPC6 (131), and CaV2.2 (132), whereas some others such as Kv4.2 appear to traffic independently of Rab11 (133). Further studies are needed to assess the role(s) of these and other trafficking proteins in mediating the surface expression of K_ATP channels from the ERC.

Aside from endocytic recycling of K_ATP channels to the plasma membrane, there is also evidence that intracellular K_ATP channels traffic to late endosomal/lysosomal compartments marked by LAMP2 (68). The authors suggested that the late endosomes may be a reservoir for recycling K_ATP channels to the membrane. The role of this recycling pathway and trafficking proteins that regulate late endosomal recycling, such as LRRK2 (134), Rab7 that regulates transport from early to late endocytic compartments (135) or Rab9 that regulates transport from late endosomes back to the trans Golgi network (136), remain to be elucidated.

Role of the Cytoskeleton

The cytoskeleton is a key component of ion channel expression and endocytic recycling. Newly synthesized channels, transporters, and pumps exit the endoplasmic reticulum and, with the assistance of molecular motors, travel along tracks formed by actin or microtubules to the plasma membrane and between trafficking compartments inside the cell. The reciprocal regulation of ion channels and the cytoskeleton has been reviewed (137). The actin cytoskeleton is often studied using compounds such as the fungal metabolite cytochalasin that inhibits growth and depolymerizes actin by binding to the actin barbed ends, or by the mushroom poison phalloidin that stabilizes filamentous (f) actin (138). Actin-destabilizing compounds lead to the activation of Na⁺ channels but they frequently suppress the activity of K⁺ channels (139). A well-described example of a K⁺ channel protein that interacts with the actin cytoskeleton by association with α-actinin is Kv1.5, which forms a voltage-activated delayed rectifier K⁺ channel. In mammalian cell lines, Kv1.5 coimmunoprecipitates and colocalize with α-actinin, whereas cytochalasin D inhibits Kv1.5 currents. Treating K_ATP channels in inside-out patches from ventricular myocytes with deoxyribonuclease (DNase) I (which forms complexes with G actin and prevents actin filament formation) or with cytochalasin D, antagonizes sulfonylurea-induced inhibition of K_ATP channels, causes a loss of ATP sensitivity, and induces a progressive loss of channel activity that can be reversed with f-actin (140, 141). Disruption of sulfonylurea binding by actin-disrupting compounds was observed in rat glomeruli and aortic rings (142, 143). A direct role for actin was demonstrated in the process by which leptin activates K_ATP channels in pancreatic β-cells (16, 17). Intracellular dialysis with the actin filament stabilizer phalloidin or treating cells with the actin-stabilizing agent jasplakinolide prevented K_ATP channel activation or trafficking by leptin. Moreover, the actin filament destabilizing agents DNase I, cytochalasin B, or latrunculin B increased K_ATP channel activity by increasing channel surface expression, whereas anti-microtubule agents nocodazole and colchicine had no effect on K_ATP channel activity. Since leptin modulated actin dynamics, the conclusion was reached that leptin activates K_ATP channels by disrupting the actin cytoskeleton (16, 17). A role for the AMPK/Rac-dependent cytoskeletal remodeling and the myosin II motor protein was demonstrated to be key in promoting K_ATP channel translocation to the plasma membrane in pancreatic β-cells (19). Overall, these data demonstrate that the actin cytoskeleton can have effects on channel opening, ATP-sensitivity, and surface trafficking, while microtubules appear to have less of a role.

Anchoring Mechanisms

The persistence of proteins at the plasmalemma can be enhanced by being localized in stable subdomains, such as caveolae, or their binding to perimembrane stable structures, such as the cortical cytoskeleton (Fig. 5).

Caveolae are small (50–100 nm) raft-like invaginations of the plasma membrane that are enriched in cholesterol and sphingolipids and are inherently stable membrane structures (49). Many ion channels, transporters, pumps, and G-protein coupled receptors localize to caveolae (47, 144). K_ATP channels can also be found in caveolae. Evidence includes the finding that K_ATP channel subunits co-fractionate with caveolar proteins (such as caveolins) after density gradient centrifugation of membrane proteins under detergent-free conditions (48, 145–147). Disrupting caveolar structure by cholesterol depletion (e.g., with methyl-β-cyclodextrin) reduces the ability of receptor signaling to affect K_ATP channel function (145–147) (see Receptor Signaling Pathways). K_ATP channels have been proposed to colocalize with, and directly interact with caveolins (147–149), which is expected to contribute to stabilizing K_ATP channels in these membrane microdomains. Caveolae are known to be stabilized by some of the C-terminal Eps15 homology domain-containing proteins, such as EHD2 (150). Indeed, EHD2 participates in K_ATP channel surface stability since a dominant negative EHD2 protein increases surface motility of K_ATP channels, leads to rapid internalization and a decrease in K_ATP channel surface density (48). In cardiomyocytes, caveolae are generally thought to be present at the mouth of t-tubules and/or the crests between t-tubules (24) and they are likely to be partially responsible for targeting/anchoring K_ATP channels to these subcellular regions.

Ankyrins are intracellular perimembrane scaffolding proteins that anchor many membrane proteins, including channels, transporters, and pumps, to the actin/spectrin cytoskeleton. The result is that the membrane proteins are targeted to specific subcellular microdomains as required for cell function (151). Ankyrin B (Ank-B), for example coordinates the expression of the Na⁺/K⁺ pump, the Na⁺/Ca²⁺ exchanger, and the inositol (1,4,5)-trisphosphate (IP₃) receptor to T-tubules in cardiac myocytes (152). K_ATP channels may be targeted to the similar domains due to their interaction with Ank-B. Evidence for a role of Ank-B in K_ATP channel targeting comes from the findings that Kir6.2 interacts with Ank-B binding domain in GST pulldown experiments and that Ank-B knockout mice or Ank-B disease-causing mutations leads to altered K_ATP channel expression (153). Ankyrin G also coordinate membrane expression of Na⁺ channels, the Na⁺/K⁺ pump, and KCNQ channels (154–157). A key role for Ank-G was demonstrated for protein targeting and interactions of Cx43, PKP2, and Nav1.5 at the cardiac intercalated disk (158–160). Evidence that K_ATP channels are targeted to the ICD, in part by being anchored to Ank-G, includes the findings that peptides against an ankyrin binding domain of Kir6.2 decreases the amplitude of a subpopulation of K_ATP channels at the ICD but not at lateral membranes, and that both Nav1.5 and Kir6.2 colocalize with Ank-G at the ICD and co-clusters, as determined by super-resolution techniques (61). Thus, Ank-B and Ank-G may have distinct roles to anchor K_ATP channels to specific regions in cardiac myocytes. These anchoring mechanisms of K_ATP channels in other tissues remain to be explored, such as the Ank-G-mediated anchoring of Na⁺ channels to the initial segment of the axon (155).

Clustering

Clustering of ion channels modulate physiological processes such as excitation-contraction coupling, regulation of vascular tone, and modulating conduction velocity. For example, clustering of ORAI channels, L-type Ca²⁺ channels, ryanodine receptors, or IP₃ receptors is responsible for producing transient, localized Ca²⁺ events (named sparks, puffs, sparklets, or microdomains), that summate to underlie global Ca²⁺ changes in cells (161, 162). Na⁺ channels also cluster in specialized subcellular regions of neurons, such as the axon initial segment, node of Ranvier, axonal terminals, postsynaptic membranes of dendrites and muscle fibers, and premyelinated axons (163) and at the ICD of cardiac myocytes (164). K_ATP channels also cluster in cardiomyocytes, particularly at the ICD, as evidenced by the large variability in currents measured in cell-attached or excised patches (51) and by direct observation of K_ATP clusters at the ICD with super-resolution methods (51, 61). One of the functional consequences of clustering is that it results in cooperative gating of some types of channels, such as L-type Ca²⁺ channels, Na⁺ channels, several types of K⁺ channels, ryanodine receptors, and IP₃ receptors (165). Another is that close juxtapositioning of different types of channels leads to functional interaction, which is best illustrated by the well-described interaction of L-type Ca²⁺ channels and RYR2 to mediate Ca²⁺-induced Ca²⁺ release in cardiac myocytes. The functional relevance of K_ATP channel clustering remains to be elucidated, but their close association with glycolytic enzymes, Na⁺ channels, and the Na⁺/K⁺ pump suggests a role in local ionic control mechanisms, possibly linked via changes in local ATP/ADP concentrations (166).

Receptor Signaling Pathways

Regulation of K_ATP channel activity by receptor signaling pathways is well described and will not be reviewed here (20). Receptor signaling pathways may also regulate K_ATP channel trafficking and endocytic recycling, but data are sparse and in some cases contradictory. CaMKII signaling has already been discussed briefly as mediating internalization in cardiac myocytes (see Dynamic changes in surface expression during pathophysiological conditions). CaMKII activation, for example during cardiac ischemia or heart failure (4, 64), appears to lead to phosphorylation of Kir6.2 at T180 and T224, which in turn enhances the binding of Kir6.2 to the μ2 subunit of the AP2 adaptor complex, thereby promoting clathrin-dependent endocytosis (66).

Glucose-induced alterations in K_ATP channel surface expression have been discussed (see Glucose-Induced Trafficking in the Pancreatic β-Cell). In pancreatic β-cells, leptin similarly increases K_ATP surface expression, by activating AMPK (Fig. 2). One study suggests that AMPK activation by leptin is mediated by Ca²⁺ influx through TRPC4 channels, which stimulates CaMKKβ to phosphorylate AMPK (18). However, other recent studies have implicated NMDA receptors as mediators of AMPK activation by leptin (167–169). In this case, leptin was shown to activate Src, which phosphorylates NMDA receptors to potentiate their activity, resulting in increased Ca²⁺ influx, activation of CaMKKβ, and phosphorylation of AMPK. Downstream of AMPK, F-actin depolymerization underlies the surface trafficking of K_ATP channels, which has been reported to be caused by AMPK-induced activation of Rac GTPase and the phosphorylation of myosin regulatory light chain (MRLC) (19). F-actin depolymerization following the leptin-AMPK signaling axis is dependent on PKA whose activity increases in response to leptin or the AMPK activator AICAR. Interestingly, knockdown of the PKA-anchoring protein AKAP79/150 prevents leptin-induced K_ATP channel trafficking, suggesting leptin signaling may lead to localized PKA activation to control actin remodeling and channel trafficking.

In cardiomyocytes, AMPK signaling has also been proposed to increase K_ATP channel surface expression (Fig. 4) particularly during ischemic preconditioning (65, 170). A key role for PKC activation has also been identified in heart cells. Ischemia, for example, causes a CaMKII-dependent internalization of K_ATP channels. Re-expression of surface K_ATP channels takes place during ischemic preconditioning, which is mimicked by adenosine and mitigated by PKC inhibitors (4). Thus, PKC signaling during preconditioning promotes K_ATP channel surface expression. PKC activation during preconditioning may well be upstream of AMPK activation, as has been demonstrated for GLUT4 upregulation in the late phase of cardioprotection (171). PKC was also shown to be involved in the upregulation of K_ATP channel currents by isoflurane-induced preconditioning (172). The signaling cascade during IPC is complex and may consist of a PKC/AMPK/p38 MAPK cascade (65). Activation by PKC with phorbol esters in heart cells also led to upregulation of surface K_ATP channel density, but curiously only in female rats (173). In heterologous expression systems, however, phorbol esters limit K_ATP channel surface expression and may divert the K_ATP channel to lysosomal degradation (68, 99, 174, 175). The underlying reasons for this difference are not clear at present and may involve participation of different PKC isoforms and/or the ability of PKC activation to signal via the AMPK/p38 MAPK axis in the non-native environment of a heterologous expression system.

Sorting and Anchoring Motifs in K_ATP Channel Subunits

Sorting of transmembrane proteins to various intracellular organelles and compartments is governed by a multitude of trafficking steps and is highly cargo specific. This specificity is imparted primarily by signals present within the cytosolic domains of the cargo proteins. These signals generally consist of short, linear sequences of amino acid residues, called motifs. For an excellent treatise of these sorting signals, the reader is referred to a comprehensive review on this topic (176). Some sorting motifs on K_ATP channel subunits have already been discussed and will not be repeated here. These include ER retention and exit motifs in Kir6.x and SURx subunits (see Folding and Assembly in the Endoplasmic Reticulum); see also Fig. 1A. Internalization signals include tyrosine-based motifs [YXXθ, with θ denoting any hydrophobic amino acid (ILMVF)] (176).

Although tyrosine-based motifs have been demonstrated to be involved in endocytosis, it should be noted that these motifs can also have roles in sorting at endosomal-lysosomal organelles and cytotoxic granules (176). Kir6.2 contains two C-terminal tyrosine-based motifs (²⁵⁸YhvI²⁶¹ and ³³⁰YskF³³³). One study demonstrated that internalization of channels can occur with the Y330C or F333I mutations, that these mutations led to an overall reduction surface expression and may be involved in neonatal diabetes by reducing ATP sensitivity (116). Another study showed that mutagenesis of the first of these (Y258A) did not affect endocytosis of Kir6.2, whereas mutagenesis of the second motif (Y330A) prevented internalization and doubled K_ATP channel surface expression (115). Internalization of K_ATP channels following CaMKII activation was also found to require an intact ³³⁰YskF³³³ motif (66). A key role for this tyrosine-based internalization motif is further suggested in patients with a genetic form of neonatal diabetes that harbor a missense variants causing amino acid mutations Y330C and F333I in Kir6.2 that inhibit spontaneous K_ATP channel endocytosis and result in a doubling of surface channels (115). The tetrapeptide tyrosine-based motif in the membrane protein coordinates its binding to the µ2 subunit of the AP2 complex, which is an important selection step during the early phases of the endocytic vesicle development. Molecular modeling of the Kir6.2 subunit has predicted that the ²⁵⁸YhvI²⁶¹ motif is hidden due to protein folding and that ³³⁰YskF³³³ is exposed (115). Molecular modeling methods have further predicted that the Kir6.2 ³³⁰YskF³³³ motif directly interacts with the AP2 complex µ2 subunit (66). Currently, there is no cryoEM structures of the K_ATP channel in complex with µ2 subunit. In recent cryoEM structures, the YSKF motif is involved in ATP binding (80). The µ2 docking needs to be revisited using available K_ATP channel cryoEM structures. The Kir6.2 ³³⁰YskF³³³ motif is fully conserved across species as well as in Kir6.1 subunits, but its role in Kir6.1-containing smooth muscle and pericyte K_ATP channels has not been explored. SUR2A contains 13 tyrosine-based motifs, but these have also not been studied.

Dileucine internalization motifs can consist minimally of adjacent Leu residues (LL). Kir6.2 contains no fewer than 28 such LL motifs and SUR2A has even more. It is extremely unlikely that each of these govern internalization. More commonly, dileucine motifs take the form of acidic cluster-dileucine motifs [DE]XXXL[LI] or DXXLL. Kir6.2 has a single C-terminal consensus sequence that corresponds to both of these forms, namely ³⁵¹EdhsLL³⁵⁶ or ³⁵²DhsLL³⁵⁶. Both Kir6.1 and SUR2A lack these dileucine motifs. Whether this Kir6.2 dileucine motif contributes to internalization is questionable. An initial study demonstrated that mutagenesis of the dileucine motif (³⁵⁶LL³⁵⁸) prevented decreased Kir6.2 surface density following PKC activation, either when co-expressed with SUR1 or SUR2, which was interpreted to mean that PKC-induced endocytosis of Kir6.2 was dependent on the dileucine motif (174). Another study confirmed that mutagenesis of ³⁵⁶LL³⁵⁸ led to a doubling of K_ATP channel surface expression, but failed to observe a decrease in the rate of endocytosis (115). The possibility was raised that this dileucine motif affects the rate of recycling to the plasma membrane, but this issue remains to be addressed experimentally. Dileucine motifs have been implicated in subcellular trafficking events other than internalization, including sorting from the trans-Golgi to the endosomal/lysosomal compartments, and mutagenesis of these dileucine motifs led to increased plasmalemmal expression of other membrane proteins as well (177–179). Mutagenesis of a dileucine pair in the distal SUR1 C-terminus (¹⁵⁶⁶LL¹⁵⁶⁷) impaired surface expression (102). Curiously, however, the effect was restricted to only the first of these Leu residues since the L1567A mutation did not have the same impact on surface expression. One interpretation of this result is that L1566 in SUR1 is a part of an anterograde sorting motif that remains to be identified.

There are other sorting motifs to consider as well. For example, NPXY or NXXY motifs regulate endocytic processing of several membrane proteins, including the LDL receptor, integrin β-1, insulin receptors and the epidermal growth factor (EGF) receptor. These motifs may coordinate binding to the membrane protein to the AP2 complex, or the Phox-homology (PX) domain-containing proteins sorting nexins, SNX17, SNX27, and SNX31 (180). Kir6 subunits lack these motifs. SUR2x contains a single NXXY motif (¹²⁰⁶NiaY¹²⁰⁹), which is not present in SUR1. Since SUR2-containing channels are endocytosed more rapidly than SUR1 channels (68), it may be worth investigating whether this sorting motif has a role.

Aside from sorting motifs, one also has to consider anchoring motifs, which can stabilize expression of membrane proteins and thereby affect surface expression and/or subcellular localization/targeting. For example, an ankyrin-binding motif (VPIALGESD) in the intracellular loop between domains II and III of Nav1.2 was found to be responsible for binding to ankyrin G (Ank-G), leading to clustering of the channel at the axonal initial segment (181). This sequence is relatively well conserved in other Na⁺ channels, including Nav1.5, where the VPIAVAESD sequence was shown to mediate binding of Nav1.5 to ankyrin G (156). K_ATP channels also have an ankyrin binding motif. Pulldown assays have demonstrated that Kir6.2 interacts with a GST-fusion protein that contains the ankyrin B (AnkB) membrane-binding domain (MBD), but not with AnkG-MBD (45). A motif in the C-terminus of Kir6.2 (³¹⁶VPI-VAEED³²³), almost identical in sequence to the Nav1.5 AnkG binding motif, mediated this interaction (153). In ventricular myocytes, AnkB is localized at lateral membranes (182) and predominantly colocalizes with the Z-line marker protein α-actinin, whereas a minor AnkB-212 isoform co-localizes only with the M-line resident protein, myomesin (46). The finding that Kir6.2 can be anchored by AnkB can therefore partly explain anchoring of K_ATP channels in lateral membranes and T-tubules of cardiac ventricular myocytes.

Kir6.2 also interacts with AnkG, apparently via the same ³¹⁶VPIVAEED³²³ domain. Overexpression of Kir6.2/SUR2A and Nav1.5 reciprocally inhibit each other electrophysiologically and reduce each other’s surface expression (without changing the total protein content) (61). Mutating the putative AnkG binding domain prevents this reciprocal functional interaction. A functional K_ATP channel is not needed since a pore-mutant nonconducting Kir6.2 subunit behaved similar to wild-type. Kir6.1, which differs from Kir6.2 by three amino acids in this region (³²⁵VSIVTEEE³³²) failed to affect Nav1.5 currents. Peptides corresponding to the Nav1.5 and Kir6.2 domains both affected Na⁺ and K_ATP channels at the ICD region, but not lateral regions, of cardiomyocytes (61). These observations, coupled with the fact that AnkG is predominantly, but not exclusively, expressed at the ICD (156, 183) support the concept that the ankyrin binding domain anchors both Na⁺ channels and K_ATP channels at the ICD (51, 61, 183).

PERSPECTIVES

Maintaining the normal level of surface K_ATP channels has been demonstrated to be of great importance for controlled insulin secretion and cardioprotection. The surface expression of K_ATP channels is regulated by a fine balance between synthesis, assembly, anterograde trafficking, membrane anchoring, endocytosis, endocytic recycling, and degradation. The mechanisms of these trafficking steps are still poorly understood. K_ATP channels express widely across different cells and tissues. K_ATP channel trafficking is mainly investigated in pancreatic β-cells, followed by cardiomyocytes and smooth muscle cells. More studies are needed to explore the trafficking of the K_ATP channels in neurons and immune cells. This knowledge is critically important for future efforts to design more effective drugs targeting the trafficking of K_ATP channels.

GRANTS

This study was supported by National Institutes of Health Grants HL146514 (to W.A.C.), HL148609 (to W.A.C. and M.D.), DA051876 (to W.A.C.), and DK066485 and DK057699 (to S.L.S.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

This article is part of the special collection “Inward Rectifying K⁺ Channels.” Jerod Denton, PhD, and Eric Delpire, PhD, served as Guest Editors of this collection.

AUTHOR CONTRIBUTIONS

H.-Q.Y., A.E., S.-L.S., and W.A.C. prepared figures; H.-Q.Y., F.A.E., A.E., and W.A.C. drafted manuscript; H.-Q.Y., F.A.E., A.E., S.A.A., N.S., T.C., M.D., S.-L.S., and W.A.C. edited and revised manuscript; H.-Q.Y., F.A.E., A.E., I.G., S.A.A., N.S., T.C., M.D. and S.-L.S., and W.A.C. approved final version of manuscript.