Reprint from

Handbook of Experimental Pharmacology, Vol. 171

Basis and Treatment of Cardiac Arrhythmias Editors: Robert S. Kass and Colleen E. Clancy

© Springer-Verlag Berlin Heidelberg 2006 Printed in Germany. Not for Sale.

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HEP (2006) 171:349–355 © Springer-Verlag Berlin Heidelberg 2006

hERG Trafficking and Pharmacological Rescue of LQTS-2 Mutant Channels G.A. Robertson1 (u) · C.T. January2 1 Dept. of

Physiology, University of Wisconsin-Madison, 601 Science Drive, Madison WI, 53711, USA [email protected] 2 Medicine (Cardiovascular), H6/354 CSC, University of Wisconsin Medical School, 600 Highland Avenue, 53792-1618 WI, Madison, USA

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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hERG Trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Trafficking Defects and Rescue of Mutant Phenotypes . . . . . . . . . . . . .

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Therapeutic Potential for Rescue . . . . . . . . . . . . . . . . . . . . . . . . .

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Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract The human ether-a-go-go-related gene (hERG) encodes an ion channel subunit underlying I Kr , a potassium current required for the normal repolarization of ventricular cells in the human heart. Mutations in hERG cause long QT syndrome (LQTS) by disrupting I Kr , increasing cardiac excitability and, in some cases, triggering catastrophic torsades de pointes arrhythmias and sudden death. More than 200 putative disease-causing mutations in hERG have been identified in affected families to date, but the mechanisms by which these mutations cause disease are not well understood. Of the mutations studied, most disrupt protein maturation and reduce the numbers of hERG channels at the membrane. Some trafficking-defective mutants can be rescued by pharmacological agents or temperature. Here we review evidence for rescue of mutant hERG subunits expressed in heterologous systems and discuss the potential for therapeutic approaches to correcting I Kr defects associated with LQTS. Keywords K+ channel · hERG · LQTS · LQT-2 · Mutation · Channelopathies · Antiarrhythmic · Proarrhythmic · I kr · Trafficking defects · Glycosylation · Torsades de pointes · RXR · Golgi · Golgi-resident protein GM130 · G601S · N470D · R752W · F805C · Fexofenadine · hERG1b · hERG1a · Rescue · Heteromultimer

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1 Introduction The human ether-a-go-go related gene (hERG) was first identified in the human hippocampus based on its similarity to Drosophila ether-a-go-go (Warmke and Ganetzky 1994), a potassium channel gene regulating membrane excitability at the neuromuscular junction (Ganetzky and Wu 1983). A candidate-gene approach led to the identification of mutations in hERG in families with type 2 inherited long QT syndrome (LQTS-2) (Curran et al. 1995), an autosomaldominant disease associated with ventricular arrhythmias and sudden death (Roden 1993). Shortly thereafter, hERG subunits were shown to be primary constituents of cardiac I Kr channels, thus explaining the underlying cause of disease as a disruption of this repolarizing current (Sanguinetti et al. 1995; Trudeau et al. 1995). Recent evidence indicates that I Kr channels are heteromultimers (Jones et al. 2004), comprising the original subunit, now termed hERG1a, and hERG1b, a subunit encoded by an alternate transcript of the hERG gene (Lees-Miller et al. 1997; London et al. 1997). The subunits are identical except for the N-terminal region, which in hERG1b is much shorter and contains a unique stretch of 36 amino acids. To date, no hERG1b-specific mutations have been associated with LQTS-2.

2 hERG Trafficking Mutations in hERG are thought to cause disease by altering I Kr functional properties (Keating and Sanguinetti 1996) and by reducing channel number at the surface via “trafficking defects” (Delisle et al. 2004). Although only a fraction of the more than 200 potential disease-causing mutations in hERG have been analyzed, most of those studied in heterologous expression systems lead to reduced surface membrane expression of channels, lower current magnitudes, and failure of mutant subunits to exit the endoplasmic reticulum (ER) and become maturely glycosylated (Zhou et al. 1998a, 1999; Furutani et al. 1999; Ficker et al. 2000a,b; January et al. 2000). The normal maturation process can be monitored by the appearance of two glycoforms reflecting progressive glycosylation in HEK-293 cells (Zhou et al. 1998b; Gong et al. 2002). hERG channels are initially core-glycosylated in the ER, producing a 135-kDa band on Western blots that is reduced in size by endoglycosidase (Endo) H. Additional glycosylation takes place in the Golgi, rendering the species that appear as the mature, Endo H resistant 155-kDa band on Western blots. The time course of maturation can be measured by pulsechase metabolic labeling using 35S and observing the time course of appearance of the 155-kDa band captured on a phosphoimager (Gong et al. 2002). At 37°C, channels reach maturity in about 24 h. As virtually all the mature band

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visible on a Western blot is sensitive to degradation by extracellularly applied proteases, such as proteinase K (Zhou et al. 1998a), transport from the Golgi to the plasma membrane must be very rapid. Many LQTS-2 mutants expressed heterologously are characterized by an abundance of the lower, immature band with little or no protein maturation. In contrast to wildtype channels, which exhibit prominent immunostaining at the membrane, the mutant channels accumulate in the ER (Zhou et al. 1998a, 1999; Ficker et al. 2000c). Although we can measure the maturation that reports the arrival of hERG subunits to the Golgi apparatus, we know little about the interactions that characterize their travels along the way. The hERG carboxy terminus carries an arginine-rich signal (RXR) that causes the subunits to be retained in the ER, but so far this is known to operate only when downstream sequences are truncated, thus presumably exposing the RXR to the ER retrieval machinery (Kupershmidt et al. 1998). How or whether the RXR sequence functions in normal hERG trafficking is unknown, but it is reasonable to hypothesize that there is an interaction with the coat protein I (COPI) machinery responsible for retrieval of misfolded or non-oligomerized subunits escaping from the ER. In ATP-gated potassium (KATP) channels, the RXR motif together with a neighboring phosphorylation site serves as a binding site for COPI, and also for 14-3-3 γ, ζ, and ε isoforms expressed in the heart. The 14-3-3 proteins compete with COPI proteins for the RXR binding site, but only when the subunits are phosphorylated and oligomerized (Yuan et al. 2003). By detecting the multimeric state of the KATP subunits, 14-3-3 thus competes with COPI for the complex and promotes its exit from the ER. hERG is known to interact with 14-3-3, though studies to date have focused on interactions mediating functional effects at the plasma membrane (Kagan et al. 2002). Upon entry to the Golgi, hERG interacts with the Golgi-resident protein GM130 (Roti Roti et al. 2002). Anchored to the Golgi membrane by an interaction with GRASP-65, GM130 tethers COPII vesicles arriving from the ER-Golgi intermediate compartment (ERGIC) via an interaction with p115 (Nakamura et al. 1997; Marra et al. 2001; Moyer et al. 2001). GM130 co-immunoprecipitates with both immature and mature hERG, suggesting it may accompany hERG from the cis to the medial Golgi, where the final glycosylation marking maturation occurs. Overexpression of GM130 in Xenopus oocytes reduces hERG current amplitude, consistent with a role as a trafficking checkpoint (Roti Roti et al. 2002). Further characterization of GM130’s role in hERG trafficking is currently under way.

3 Trafficking Defects and Rescue of Mutant Phenotypes The defects underlying the failure of LQTS-2 mutants to mature are poorly understood. LQTS-2 mutations are found throughout the hERG protein, including

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the cytosolic amino terminus, the transmembrane domains, and throughout the long, cytosolic carboxy terminus (Delisle et al. 2004). Possible mechanisms preventing maturation include folding or assembly defects, failure to be appropriately processed in the Golgi, loss of checkpoint protein interactions, or mistargeting to degradative pathways rather than to the plasma membrane (Ellgaard and Helenius 2003). Mutations with a dominant-negative phenotype may cause protein misfolding but do not disrupt oligomerization with wildtype subunits, which are rendered dysfunctional by association with the mutants. In contrast, loss-of-function mutations, may signal defects in oligomerization, as wildtype subunits form functional channels unhindered by coexpressed mutant subunits. Both classes of mutant proteins are unlikely to proceed beyond the ER, following instead an expedited path to degradation. Perhaps surprisingly, our understanding of these underlying defects may be illuminated by the even more mysterious phenomenon of rescue. The plasma membrane expression of some LQTS-2 mutants in heterologous systems can be restored by reducing temperature or applying hERG channel blockers (Zhou et al. 1999). Other compounds, such as fexofenadine (Rajamani et al. 2002), a derivative of the hERG blocker terfenadine (Suessbrich et al. 1996), and thapsigargin (Delisle et al. 2003), a calcium pump inhibitor that diminishes calcium-dependent chaperone protein activity, have also been shown to rescue LQTS-2 mutations. Each of these interventions likely mediates rescue by a different mechanism. Reduced temperature is thought to stabilize folding intermediates, whereas channel blockers, which bind to the internal pore vestibule where the four subunits interact, may stabilize oligomeric integrity. Thapsigargin inhibits the sarcoplasmic/ER Ca++ -ATPase, resulting in a reduced lumenal Ca++ concentration in the ER (Inesi and Sagara 1992). For mutant cystic fibrosis transmembrane regulator (CFTR) channels, it has been proposed that Ca++ -dependent chaperones, which handcuff improperly folded proteins while they await degradation, lose their grip as Ca++ levels drop and allow the errant channels to escape to the plasma membrane (Egan et al. 2002; Delisle et al. 2003). At least four hERG mutations, G601S, N470D, R752W and F805C can be rescued by reduced temperature, consistent with folding defects (Zhou et al. 1999; Ficker et al. 2000c; Delisle et al. 2003). G601S and R752W subunits exhibit enhanced binding to the chaperone proteins Hc70 and Hsp90, accompanied by an increase in degradation, suggesting the mutants cannot be coaxed by the normal, physiological mechanisms into the correct conformation for export (Ficker et al. 2003). G601S and F805C can be rescued by thapsigargin but not other inhibitors of the sarcoplasmic/ER Ca++ -ATPase, suggesting a mechanism distinct from that for CFTR mutant rescue (Delisle et al. 2003). Interestingly, of these three mutants, G601S is perhaps the most compliant of all, as it is rescued by all approaches utilized so far. In contrast, F805C is rescued only by temperature and thapsigargin (Delisle et al. 2003), and N470D by temperature and pore blockers (Zhou et al. 1999; Rajamani et al. 2002).

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Thus, even among mutants characterized as folding-defective, the molecular mechanisms of disease must be quite diverse. R752W is unlikely even to form oligomers, as it exhibits a loss-of-function rather than a dominant-negative phenotype (Ficker et al. 2003), whereas G601S and N470D oligomerize effectively and respond to the stabilizing effects of reduced temperature on folding or the binding of drugs to the pore (Zhou et al. 1999; Rajamani et al. 2002), which may reinforce the oligomeric structure required for ER export.

4 Therapeutic Potential for Rescue Pharmacological or chemical rescue strategies have a therapeutic potential only if the rescued I Kr channels are sufficiently functional to support normal cardiac repolarization. Most examples of rescue have occurred with hERG channel blockers, which carry the risk for acquired LQTS. This is not so for fexofenadine, a derivative of the hERG blocker terfenadine. Fexofenadine mediates rescue of G601S and N470D at an IC50 300-fold lower than that for drug block, indicating for the first time that rescue and restoration of normal I Kr function can potentially be decoupled from the risk for LQTS and torsades de pointes (Rajamani et al. 2002). At the surface, rescued G601S and N470D channels exhibit normal gating and permeation (Furutani et al. 1999; Zhou et al. 1999).

5 Conclusions Recent advances indicate that hERG channels with LQTS mutations may be rescued pharmacologically, opening the door for therapeutic intervention in the disease process. One compound, fexofenadine, can rescue certain mutants without the deleterious effects of channel block and associated risk of acquired LQTS. Mutants with relatively mild folding defects are likely the best candidates for rescue, as they seem to function normally upon reaching the plasma membrane. These studies underscore the importance of determining the specific mutation carried by a patient and evaluating the corresponding mutant phenotype and its receptiveness to rescue in heterologous systems. There is also a need to understand in greater detail the mechanisms of hERG subunit folding and assembly, as well as the protein–protein interactions in the trafficking pathway. Disruption of any of these events may lead to disease, and all represent potential targets for therapeutic rescue. Heterologous expression systems used to evaluare LQTS mutants should incorporate wildtype subunits as well as hERG1b subunits to better mimic native I Kr channels. Mutations introduced into heteromeric hERG1a/1b channels may confer different mutant

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phenotypes and responses to rescue agents compared with hERG1a homomeric mutant channels. Ultimately, this information will contribute to a rational and personalized approach to therapeutic treatment of patients with long QT syndrome.

References Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, Keating MT (1995) A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80:795–803 Delisle BP, Anderson CL, Balijepalli RC, Anson BD, Kamp TJ, January CT (2003) Thapsigargin selectively rescues the trafficking defective LQT2 channels G601S and F805C. J Biol Chem 278:35749–35754 Delisle BP, Anson BD, Rajamani S, January CT (2004) Biology of cardiac arrhythmias: ion channel protein trafficking. Circ Res 94:1418–1428 Egan ME, Glockner-Pagel J, Ambrose C, Cahill PA, Pappoe L, Balamuth N, Cho E, Canny S, Wagner CA, Geibel J, Caplan MJ (2002) Calcium-pump inhibitors induce functional surface expression of Delta F508-CFTR protein in cystic fibrosis epithelial cells. Nat Med 8:485–492 Ellgaard L, Helenius A (2003) Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4:181–191 Ficker E, Dennis AT, Obejero-Paz CA, Castaldo P, Taglialatela M, Brown AM (2000a) Retention in the endoplasmic reticulum as a mechanism of dominant-negative current suppression in human long QT syndrome. J Mol Cell Cardiol 32:2327–2337 Ficker E, Thomas D, Viswanathan PC, Dennis AT, Priori SG, Napolitano C, Memmi M, Wible BA, Kaufman ES, Iyengar S, Schwartz PJ, Rudy Y, Brown AM (2000b) Novel characteristics of a misprocessed mutant HERG channel linked to hereditary long QT syndrome. Am J Physiol Heart Circ Physiol 279:H1748–1756 Ficker E, Dennis AT, Wang L, Brown AM (2003) Role of the cytosolic chaperones Hsp70 and Hsp90 in maturation of the cardiac potassium channel HERG. Circ Res 92:e87–100 Ficker EK, Thomas D, Viswanathan P, Rudy Y, Brown AM (2000c) Rescue of a misprocessed mutant HERG channel linked to hereditary long QT syndrome. Biophys J 78:342A Furutani M, Trudeau MC, Hagiwara N, Seki A, Gong Q, Zhou Z, Imamura S, Nagashima H, Kasanuki H, Takao A, Momma K, January CT, Robertson GA, Matsuoka R (1999) Novel mechanism associated with an inherited cardiac arrhythmia: defective protein trafficking by the mutant HERG (G601S) potassium channel. Circulation 99:2290–2294 Gong Q, Anderson CL, January CT, Zhou Z (2002) Role of glycosylation in cell surface expression and stability of HERG potassium channels. Am J Physiol Heart Circ Physiol 283:H77–84 Inesi G, Sagara Y (1992) Thapsigargin, a high affinity and global inhibitor of intracellular Ca2+ transport ATPases. Arch Biochem Biophys 298:313–317 January CT, Gong Q, Zhou Z (2000) Long QT syndrome: cellular basis and arrhythmia mechanism in LQT2. J Cardiovasc Electrophysiol 11:1413–1418 Jones EM, Roti Roti EC, Wang J, Delfosse SA, Robertson GA (2004) Cardiac IKr channels minimally comprise hERG 1a and 1b subunits. J Biol Chem 279:44690–44694 Kagan A, Melman YF, Krumerman A, McDonald TV (2002) 14-3-3 amplifies and prolongs adrenergic stimulation of HERG K+ channel activity. EMBO J 21:1889–1898 Keating MT, Sanguinetti MC (1996) Molecular genetic insights into cardiovascular disease. Science 272:681–685

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Kupershmidt S, Snyders DJ, Raes A, Roden DM (1998) A K+ channel splice variant common in human heart lacks a C-terminal domain required for expression of rapidly activating delayed rectifier current. J Biol Chem 273:27231–27235 Lees-Miller JP, Kondo C, Wang L, Duff HJ (1997) Electrophysiological characterization of an alternatively processed ERG K+ channel in mouse and human hearts. Circ Res 81:719–726 London B, Trudeau MC, Newton KP, Beyer AK, Copeland NG, Gilbert DJ, Jenkins NA, Satler CA, Robertson GA (1997) Two isoforms of the mouse ether-a-go-go-related gene coassemble to form channels with properties similar to the rapidly activating component of the cardiac delayed rectifier K+ current. Circ Res 81:870–878 Marra P, Maffucci T, Daniele T, Tullio GD, Ikehara Y, Chan EKL, Luini A, Beznoussenko G, Mironov A, DeMatteis MA (2001) The GM130 and GRASP65 golgi proteins cycle through and define a subdomain of the intermediate compartment. Nat Cell Biol 3:1101–1114 Moyer BD, Allan BB, Balch WE (2001) Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis-Golgi tethering. Traffic 2:268–276 Nakamura N, Lowe M, Levine TP, Rabouille C, Warren G (1997) The vesicle docking protein p115 binds GM130, a cis-Golgi matrix protein, in a mitotically regulated manner. Cell 89:445–455 Rajamani S, Anderson CL, Anson BD, January CT (2002) Pharmacological rescue of human K(+) channel long-QT2 mutations: human ether-a-go-go-related gene rescue without block. Circulation 105:2830–2835 Roden DM (1993) Torsade de pointes. Clin Cardiol 16:683–686 Roti Roti EC, Myers CD, Ayers RA, Boatman DE, Delfosse SA, Chan EK, Ackerman MJ, January CT, Robertson GA (2002) Interaction with GM130 during HERG ion channel trafficking. Disruption by type 2 congenital long QT syndrome mutations. J Biol Chem 277:47779–47785 Sanguinetti MC, Jiang C, Curran ME, Keating MT (1995) A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81:299–307 Suessbrich H, Waldegger S, Lang F, Busch AE (1996) Blockade of HERG channels expressed in Xenopus oocytes by the histamine receptor antagonists terfenadine and astemizole. FEBS Lett 385:77–80 Trudeau MC, Warmke JW, Ganetzky B, Robertson GA (1995) HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269:92–95 Warmke JW, Ganetzky B (1994) A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci U S A 91:3438–3442 Yuan H, Michelsen K, Schwappach B (2003) 14-3-3 dimers probe the assembly status of multimeric membrane proteins. Curr Biol 13:638–646 Zhou Z, Gong Q, Epstein ML, January CT (1998a) HERG channel dysfunction in human long QT syndrome. Intracellular transport and functional defects. J Biol Chem 273:21061– 21066 Zhou Z, Gong Q, January CT (1999) Correction of defective protein trafficking of a mutant HERG potassium channel in human long QT syndrome. Pharmacological and temperature effects. J Biol Chem 274:31123–31126