We have established stably transfected HEK 293 cell lines expressing high levels of functional human ether-a go-go-related gene (HERG) channels. We used these cells to study biochemical characteristics of HERG protein, and to study electrophysiological and pharmacological properties of HERG channel current at 35 degrees C. HERG-transfected cells expressed an mRNA band at 4.0 kb. Western blot analysis showed two protein bands (155 and 135 kDa) slightly larger than the predicted molecular mass (127 kDa). Treatment with N-glycosidase F converted both bands to smaller molecular mass, suggesting that both are glycosylated, but at different levels. HERG current activated at voltages positive to -50 mV, maximum current was reached with depolarizing steps to -10 mV, and the current amplitude declined at more positive voltages, similar to HERG channel current expressed in other heterologous systems. Current density at 35 degrees C, compared with 23 degrees C, was increased by more than twofold to a maximum of 53.4 +/- 6.5 pA/pF. Activation, inactivation, recovery from inactivation, and deactivation kinetics were rapid at 35 degrees C, and more closely resemble values reported for the rapidly activating delayed rectifier K+ current (I(Kr)) at physiological temperatures. HERG channels were highly selective for K+. When we used an action potential clamp technique, HERG current activation began shortly after the upstroke of the action potential waveform. HERG current increased during repolarization to reach a maximum amplitude during phases 2 and 3 of the cardiac action potential. HERG contributed current throughout the return of the membrane to the resting potential, and deactivation of HERG current could participate in phase 4 depolarization. HERG current was blocked by low concentrations of E-4031 (IC50 7.7 nM), a value close to that reported for I(Kr) in native cardiac myocytes. Our data support the postulate that HERG encodes a major constituent of I(Kr) and suggest that at physiological temperatures HERG contributes current throughout most of the action potential and into the postrepolarization period.

Background— The KCNH2 or human ether-a-go-go related gene ( hERG ) encodes the Kv11.1 α-subunit of the rapidly activating delayed rectifier K + current ( I Kr ) in the heart. Type 2 congenital long-QT syndrome (LQT2) results from KCNH2 mutations that cause loss of Kv11.1 channel function. Several mechanisms have been identified, including disruption of Kv11.1 channel synthesis (class 1), protein trafficking (class 2), gating (class 3), or permeation (class 4). For a few class 2 LQT2-Kv11.1 channels, it is possible to increase surface membrane expression of Kv11.1 current ( I Kv11.1 ). We tested the hypotheses that (1) most LQT2 missense mutations generate trafficking-deficient Kv11.1 channels, and (2) their trafficking-deficient phenotype can be corrected. Methods and Results— Wild-type (WT)-Kv11.1 channels and 34 missense LQT2-Kv11.1 channels were expressed in HEK293 cells. With Western blot analyses, 28 LQT2-Kv11.1 channels had a trafficking-deficient (class 2) phenotype. For the majority of these mutations, the class 2 phenotype could be corrected when cells were incubated for 24 hours at reduced temperature (27°C) or in the drugs E4031 or thapsigargin. Four of the 6 LQT2-Kv11.1 channels that had a wild-type–like trafficking phenotype did not cause loss of Kv11.1 function, which suggests that these channels are uncommon sequence variants. Conclusions— This is the first study to identify a dominant mechanism, class 2, for the loss of Kv11.1 channel function in LQT2 and to report that the class 2 phenotype for many of these mutant channels can be corrected. This suggests that if therapeutic strategies to correct protein trafficking abnormalities can be developed, it may offer clinical benefits for LQT2 patients.

Calcium channel antagonists have diverse effects on cardiac electrophysiology. We studied the effects of verapamil, diltiazem, and nifedipine on HERG K+ channels that encode IKr in native heart cells. In our experiments, verapamil caused high-affinity block of HERG current (IC50=143.0 nmol/L), a value close to those reported for verapamil block of L-type Ca2+ channels, whereas diltiazem weakly blocked HERG current (IC50=17.3 micromol/L), and nifedipine did not block HERG current. Verapamil block of HERG channels was use and frequency dependent, and verapamil unbound from HERG channels at voltages near the normal cardiac cell resting potential or with drug washout. Block of HERG current by verapamil was reduced by lowering pHO, which decreases the proportion of drug in the membrane-permeable neutral form. N-methyl-verapamil, a membrane-impermeable, permanently charged verapamil analogue, blocked HERG channels only when applied intracellularly. Verapamil antagonized dofetilide block of HERG channels, which suggests that they may share a common binding site. The C-type inactivation-deficient mutations, Ser620Thr and Ser631Ala, reduced verapamil block, which is consistent with a role for C-type inactivation in high-affinity drug block, although the Ser620Thr mutation decreased verapamil block 20-fold more than the Ser631Ala mutation. Our findings suggest that verapamil enters the cell membrane in the neutral form to act at a site within the pore accessible from the intracellular side of the cell membrane, possibly involving the serine at position 620. Thus, verapamil shares high-affinity HERG channel blocking properties with other class III antiarrhythmic drugs, and this may contribute to its antiarrhythmic mechanism.

The chromosome 7-linked form of congenital long QT syndrome (LQT2) is caused by mutations in the human ether-a-go-go-related gene (HERG) that encodes the rapidly activating delayed rectifier potassium channel. One mechanism for the loss of normal channel function in LQT2 is defective protein trafficking, which results in the failure of the channel protein to reach the plasma membrane. Here we show that the N470D LQT2 mutant protein is trafficking-deficient when expressed at 37°C in HEK293 cells, whereas at 27°C its trafficking to the plasma membrane and channel function are markedly improved. We further show that the antiarrhythmic drug E-4031, which selectively blocks HERG channels, also corrects defective protein trafficking of the N470D mutant and can restore the generation of HERG current. Similar findings were obtained with the drugs astemizole and cisapride, as well as with high concentrations of glycerol. The effect of E-4031 on HERG protein trafficking was concentration-dependent and required low drug concentrations (saturation present at 5 M), developed rapidly with drug exposure, and occurred post-translationally. These findings suggest that protein misfolding leading to defective trafficking of some HERG LQT mutations may be corrected by specific pharmacological strategies. Human congenital long QT syndrome (LQT)1 is characterized by repolarization abnormalities in the heart, leading to cardiac arrhythmias, syncope, and sudden death. Defects in genes encoding pore-forming or auxiliary subunits have been identified for three types of cardiac ion channels (1-5) in human LQT. The most common defects are found in genes encoding the KVLQT1 and HERG potassium channels (1-3). For the HERG channel, which encodes the pore-forming subunit of the cardiac rapidly activating delayed rectifier potassium channel (1, 6 -8), we have recently reported that the loss of channel function can be caused by multiple mechanisms including abnormal protein trafficking, the generation of nonfunctional channels, and altered channel gating (9). In the present experiments, we studied the LQT mutation N470D heterologously expressed in a mammalian cell line. This mutation, when expressed in Xenopus oocytes, has been reported to generate HERG current with reduced amplitude (30% of wild type) and altered voltage dependence of activation (10). We found that the protein trafficking of N470D mutant is highly temperaturesensitive. In addition, we showed that the defective protein trafficking can be corrected by drugs that selectively block HERG channels. EXPERIMENTAL PROCEDURESSite-directed Mutagenesis and Transfection-The HERG N470D mutation was generated by site-directed mutagenesis of wild type HERG cDNA using the GeneEditor in vitro mutagenesis system (Promega, Madison, WI). Stable transfection of human embryonic kidney (HEK293) cells with HERG wild type and N470D cDNA was carried out using a LipofectAMINE method (11).Cell Culture and Patch Clamp Recordings-HEK293 cells were cultured at 37°C except where noted when the temperature was ...

Mutations in HERG are associated with human chromosome 7-linked congenital long QT (LQT-2) syndrome. We used electrophysiological, biochemical, and immunohistochemical methods to study the molecular mechanisms of HERG channel dysfunction caused by LQT-2 mutations. Wild type HERG and LQT-2 mutations were studied by stable and transient expression in HEK 293 cells. We found that some mutations (Y611H and V822M) caused defects in biosynthetic processing of HERG channels with the protein retained in the endoplasmic reticulum. Other mutations (I593R and G628S) were processed similarly to wild type HERG protein, but these mutations did not produce functional channels. In contrast, the T474I mutation expressed HERG current but with altered gating properties. These findings suggest that the loss of HERG channel function in LQT-2 mutations is caused by multiple mechanisms including abnormal channel processing, the generation of nonfunctional channels, and altered channel gating.The congenital long QT syndrome is a disorder associated with delayed cardiac repolarization and prolonged electrocardiographic QT intervals and the development of ventricular arrhythmias (torsades de pointes) and sudden death (1). One cause of congenital long QT syndrome is mutation in the human ether-a-go-go-related gene (HERG) producing chromosome 7-linked congenital long QT syndrome (LQT-2) 1 (2). HERG encodes a voltage-gated potassium channel (3). HERG channel current has been shown to have properties similar to the rapidly activating delayed rectifier K ϩ current (I Kr ), and it plays an important role in cardiac action potential repolarization in the mammalian heart (4 -6). HERG channels are also an important target for block by many drugs, and suppression of HERG current causes action potential prolongation and cardiac arrhythmias (5-12). Therefore, HERG channels have emerged as an important cardiac ion channel.More than 30 HERG mutations have been identified in LQT-2 patients (2, 13-18). The electrophysiological properties of a few LQT-2 mutations have been studied in Xenopus oocytes, where they have been shown to result in reduced or absent HERG current (19). Although the molecular basis for some congenital human diseases is known to involve multiple mechanisms including defective protein processing and abnormal protein function, the molecular basis for long QT syndrome has not been studied. In the present work, we used electrophysiological, biochemical, and immunohistochemical methods to study intracellular protein processing and functional properties of wild type and five LQT-2 mutant HERG channels. Our findings show that some mutant HERG proteins are not processed to the mature form of the channel. Other mutant HERG proteins undergo normal processing but do not form functional channels, or they gate abnormally. These findings provide new information about the molecular mechanisms for the failure of mutant LQT-2 channels to generate normal HERG current. Table I were generated by site-directed mutagenesis using the Altered Site II in vi...

681oped (including cisapride, prucalopride and tegaserod) are partial agonists, but only tegaserod is presented as such in Table 1 of Dr Spiller's paper.Secondly, we would like to point out that buspirone and sumatriptan are both defined as 5-HT 1P receptor agonists throughout the review, whereas buspirone is a 5-HT 1A receptor agonist (and also a dopamine D 2 receptor antagonist) [11] and sumatriptan is the prototype 5-HT 1B/D receptor agonist [12]. Sumatriptan has been extensively investigated by Dr Tack's group [13,14] and was found to have important effects on gastric tone and perception of gastric distension [14], but the receptors involved in mediating its effects are still a matter of debate. Therefore, the fact that 5-HT 1P receptors are presented as potential therapeutic targets in functional gut disorders is misleading, all the more so because 5-HT 1P receptors are not included in the official IUPHAR classification of serotonin receptors [12]. We are aware that the presence of 5-HT 1P receptors is reported in enteric neurones [15], but the hypothesis that sumatriptan determines its gastric motor effects in vivo through these receptors still awaits confirmation by pharmacological studies with selective antagonists. It is noteworthy, however, that a preliminary report by our group [16] showed that the gastric motor effect of sumatriptan was blocked in vivo by GR127935, a selective 5-HT 1B/D receptor antagonist.

Gong, Qiuming, Corey L. Anderson, Craig T. January, and Zhengfeng Zhou. Role of glycosylation in cell surface expression and stability of HERG potassium channels. Am J Physiol Heart Circ Physiol 283: H77-H84, 2002. First published March 7, 2002 10.1152/ajpheart.00008. 2002 encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel in the heart. We previously showed that HERG channel protein is modified by N-linked glycosylation. HERG protein sequence contains two extracellular consensus sites for N-linked glycosylation (N598, N629). In this study, we used the approaches of site-directed mutagenesis and biochemical modification to inhibit N-linked glycosylation and studied the role of glycosylation in the cell surface expression and turnover of HERG channels. Our results show that N598 is the only site for N-linked glycosylation and that glycosylation is not required for the cell surface expression of functional HERG channels. In contrast, N629 is not used for glycosylation, but mutation of this site (N629Q) causes a protein trafficking defect, which results in its intracellular retention. Pulse-chase experiments show that the turnover rate of nonglycosylated HERG channel is faster than that of the glycosylated form, suggesting that N-linked glycosylation plays an important role in HERG channel stability.heart; arrhythmia; ion channels; mutations; patch clamp HUMAN ether-à -go-go-related gene (HERG) encodes a K ϩ channel that belongs to the EAG family of voltagegated ion channels (35). HERG channel current has properties similar to the rapidly activating delayed rectifier K ϩ current in the heart, which plays a central role in action potential repolarization (27,30,34,38). Mutations in HERG cause the chromosome 7-linked form of inherited long QT syndrome (LQT2) (4), and to date Ͼ100 HERG mutations have been identified in LQT2 patients (13,14,31). HERG also plays an important role in the acquired form of long QT syndrome, because most drugs that cause drug-induced long QT syndrome block HERG channels. Thus HERG is an important target in both the inherited and acquired forms of long QT syndrome.Although electrophysiological and pharmacological properties of HERG channels have been studied widely, less is known about the biochemical processing of HERG channel protein. We previously reported (37, 38) that wild-type HERG channel protein expressed in HEK293 cells exhibits two bands on Western blot analysis and that the generation of both bands involves asparagine (N)-linked glycosylation. We showed (37) that the larger-molecular-mass band is the fully glycosylated, mature form of HERG channel located in the plasma membrane and the smaller-molecular-mass band is a core-glycosylated, precursor form of HERG channel located in the endoplasmic reticulum (ER). The HERG protein sequence contains two extracellular consensus sites for N-linked glycosylation (N598, N629). Recently, Petrecca et al. (20) reported that both single (N598Q, N629Q) and double (N598Q-N629Q) mutations resulted in t...

Desmethylastemizole and astemizole cause equipotent block of HERG channels, and these are among the most potent HERG channel antagonists yet studied. Because desmethylastemizole becomes the dominant compound in serum, these findings support the postulate that it becomes the principal cause of long QT syndrome observed in patients following astemizole ingestion. Norastemizole block of HERG channels is weaker; thus, the risk of producing ventricular arrhythmias may be lower. These findings underscore the potential roles of some H1-receptor antagonist metabolites as K+ channel antagonists.