Ion Channel Trafficking in the Heart

Overview of Ion Channel Trafficking in the Heart

A remarkable aspect of cardiac ion channel biology is that individual ion channels have half-lives on the order of hours. For example, Cx43 gap junction proteins have a half-life of 1 to 3 hours, ^, and calcium channels have half-lives that are reported to be 2 to 8 hours. ^, The short life span of ion channels suggests that there needs to be efficiency in their life cycle and movements. There is precious little time for the proteins of ion channels to form, for the proteins to oligomerize into channels, for delivery of de novo channels to the correct subdomain on the plasma membrane, for functional activity once in the membrane, and finally for internalization and either degradation or reinsertion into the membrane.

Cardiomyocytes (CMs) use common intracellular organelles and machinery to produce and shuttle ion channel proteins to their specific organelles and functional subdomains at the cell membrane. After gene transcription in the nuclei, proteins are translated and subjected to posttranslational modification in the endoplasmic reticulum (ER) and then further modified in the Golgi apparatus. For ion channels, sorting and delivery to their subcellular destination begins in the Golgi complex, which is usually found adjacent to the lateral side of each nucleus in mammalian ventricular CMs. Colocalized with each Golgi is the centrosome at which microtubules are nucleated and extend throughout the cell. The sorting of proteins mainly occurs at the trans-Golgi network (TGN). Cargo proteins are sorted into post-Golgi carriers, which are docked onto molecular motors and delivered to the cell periphery along microtubules. These extending microtubules form an intricate and dynamic outgoing network capable of shuttling ion channel–containing vesicles to their destinations. In the context of trafficking, one can consider the Golgi to be the “loading dock” and the microtubules to be the “highways” along which packets of channels are delivered to the plasma membrane. The actin cytoskeleton is also involved, both serving as rest stops for channels en route to the plasma membrane and to guide microtubule growth patterns.

The mechanisms by which microtubules exert their specificity in interacting with membrane subdomains are now being elucidated. Our report in 2007 and subsequent studies have led to the targeted delivery model of ion channel delivery. The targeted delivery model has since been supported by multiple other reports.

Targeted Delivery

Targeted delivery is the understanding that channels, once formed and after exiting the Golgi, can be rapidly directed across the cytoplasm to their respective specific membrane subdomains. The highways for transport are microtubules whose negative ends originate at Golgi-oriented microtubule-organizing centers and whose positive ends are growing outward and can be captured at the plasma membrane by membrane anchor proteins and complexes. The specificity of delivery is a combination of (1) individual channels and their components; (2) the microtubule and actin cytoskeleton together with plus end–tracking proteins (+TIPs) at the positive ends of microtubules, which guide microtubule growth and capture; and (3) membrane-bound anchor complexes, which capture the microtubule, thus completing the highway for channel delivery ( Fig. 17.1 ). Targeted delivery is explained in this chapter with two different types of channels: Cx43 hemichannels to intercalated disks and Ca _V 1.2 channels to T-tubules, followed by an exploration of critical components in the machinery.

Fig. 17.1, Channel trafficking in healthy and failing hearts.

Cx43 Trafficking in the Heart

Connexins are ubiquitous transmembrane proteins that are encoded by over 20 different genes in humans, with Cx43 being the most commonly expressed in all organ systems, particularly the heart. ^, An extensively studied and well-appreciated function of connexins is their ability to form gap junctions. To form a gap junction, six connexin monomers from one cell oligomerize to form a transmembrane channel referred to as a connexon or hemichannel . The connexons from one cell then dock and couple with apposing connexons on neighboring cells and coalesce into dense gap junction plaques. Gap junctions are specialized channels that aid in the intercellular exchange of small metabolites, secondary messengers, and ions carrying electrical signals between neighboring cells, thereby allowing cells to cooperate both electrically and metabolically. ^, ^,

In the heart, the localization of Cx43 gap junctions at the intercalated disks is crucial to providing the intercellular coupling necessary for rapid action potential propagation through the myocardium and synchronized cardiac contraction. Altered Cx43 localization and losses in cell-cell gap junction coupling occur during cardiac disease and contribute to abnormal impulse propagation and arrhythmogenic substrates, leading to sudden cardiac death. In addition, a large number of studies have demonstrated a decrease in expression and/or lateralization and heterogeneous distribution of Cx43 in the myocardium of patients with hypertrophic cardiomyopathies, dilated cardiomyopathies, ^, ischemic cardiomyopathies, ^, ^, and clinical congestive heart failure. In general, altered Cx43 localization is a result of a diseased myocardium, with consequences that include lethal arrhythmias. The mislocalization of Cx43 during disease reflects impaired trafficking to the intercalated disks. Thus, to preserve rapid and organized propagation of electrical signals, it is important to focus on the mechanisms of normal Cx43 trafficking and the changes that occur with disease.

Cx43 Forward Trafficking in Normal Heart Physiology

Cx43 oligomerizes into hemichannels at the TGN, which is relatively late for such an event to occur because other connexins oligomerize in ER. This may represent a means of controlling heteromeric hemichannel formations with other connexin isoforms. On exiting the TGN, vesicles that contain Cx43 hemichannels must navigate the complex CM intracellular environment, a feat they achieve by trafficking along dynamic microtubules.

Trafficking Cx43 hemichannels to the intercalated disks involves a major plus end–binding protein, EB1, which is known to be necessary for the targeted delivery of Cx43 hemichannels to adherens junction complexes (see Fig. 17.1 ). Through interaction with another plus-end protein, p150GLUED, the EB1-tipped microtubule specifically complexes with β-catenin molecules at the fascia adherens of intercalated disks. The vesicular cargo is unloaded and subsequently inserted into the plasma membrane at nearby gap junctions. Historically, other reports proposed a less specific paradigm of connexin delivery, whereby connexons are inserted indiscriminately into the lateral membrane of the cell and freely diffuse to gap junction structures. Both models can exist in parallel. Nevertheless, the inefficiency of lateral diffusion to a few specific subdomains, the short half-life of connexins (1–3 hours in the myocardium), ^, and the complex interactions between a single cell with multiple neighboring CMs all suggest that directed targeting can be a more effective form of connexon localization to intercalated discs.

In targeted delivery, the specificity of delivery involves a dynamic cytoskeleton that arranges trafficking highways based on local cues. For instance, membrane subdomains have distinctive membrane-bound anchor proteins that capture with specificity a subgroup of microtubules. Thus channel delivery directly to membrane subdomains are determined in part by the characteristic anchor protein at the subdomain. For Cx43 delivery to the intercalated disk, EB1-tipped microtubules bind to N-cadherin. Desmoplakin may also be involved in capturing the EB1-tipped microtubule for Cx43 delivery, although the transmembrane domain still appears to be N-cadherin rather than desmosomal desmoglein. Cx43 hemichannels are not trafficking to the intercalated disc per se, but rather to N-cadherin, which happens to be at the intercalated disc.

Other than microtubules, there is an increasing appreciation for the involvement of the nonsarcomeric actin cytoskeleton in the targeted delivery of Cx43. The fundamental question remains with regard to why actin is involved in Cx43 trafficking. If vesicles that contain Cx43 can depart the Golgi and ride a microtubule highway straight to its proper subdomain, is there a need for actin filaments that appear to slow down vesicle transport? Actin can have at least two important roles in Cx43 forward delivery. The first is to help contribute specificity to delivery. Vesicles transported along microtubules on kinesin motors move rapidly at a rate of about 1 micron per second. Thus delivery to most locations in a cell membrane can occur within a minute. Association with important accessory proteins and posttranslational modification of channels, both of which can affect delivery destination, probably also happen en route between the Golgi and membrane. Hopping off the microtubule highway on an actin “rest stop,” analogous to a highway rest stop with convenience stores, could allow Cx43 and the vesicle containing it to pick up accessory proteins and enable the needed posttranslational modification. Such rest stops would occur at the Z-disk, subcortical locations, or other important cytoskeleton intersections in the cytoplasm. These actin rest stops could also allow the Cx43-containing vesicles to use multiple microtubule highways in their delivery path. The Golgi exiting the microtubule could be destined for an actin rest stop, allowing for a different membrane domain–specific microtubule to finish the delivery. At any given point in time, the majority of intracellular Cx43 channels are not moving rapidly on microtubules but rather are stationary and associated with nonsarcomeric actin, ^, supporting the possibility that actin rest stops are critical for channel processing and determination of its particular final membrane subdomain destination.

The second potential role for actin in microtubule-based forward delivery pertains to the microtubules themselves. In nonmyocyte systems, actin can help stabilize and guide microtubules. ^, Actin is the blueprint along and across which microtubule highways are patterned. In this respect, actin involvement could be upstream to microtubules in determining the location of Cx43 delivery. As discussed later, there is an intriguing possibility that smaller truncated components of the channel itself could help pattern actin, laying the blueprint for microtubule highways and, ultimately, hemichannels of full-length Cx43 hemichannels. ^,

Cx43 Forward Trafficking in Heart Pathophysiology

Smyth and colleagues have found that when isolated CMs are subjected to oxidative stress, the Cx43 gap junction delivery to intercalated disks is impaired because of the disruption of the forward trafficking machinery. Specifically, oxidative stresses cause the microtubule plus-end protein EB1 to disassociate from the tips of microtubules, impairing microtubule attachment to adherens junction structures and the subsequent delivery of Cx43 hemichannels to the plasma membrane (see Fig. 17.1 ). The manipulation of EB1 and of the upstream regulators of EB1 localization at microtubules could potentially preserve or enhance gap junction coupling during stress. Because many ion channels rely on microtubules for their transport, it is likely that such a disruption of microtubule trafficking machinery inhibits the delivery of many essential channels to the sarcolemma.

Disruption of the actin cytoskeleton is also a possible cause of altered channel trafficking in disease. Acute myocardial ischemia decreases biochemical Cx43 clustering with actin and causes a 50% decrease of Cx43 delivery to intercalated discs. Depolymerizing actin polymerization with latrunculin A causes a similar decrease in Cx43 delivery to intercalated discs, with little additional disruption by acute ischemia, suggesting that the role of actin in Cx43 delivery is highly sensitive to ischemic conditions.

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