KChannelDB: Extraction of mutation data from the literature

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This data was extracted from Medline abstracts and full texts (when available) in an automated manner.

The table below describes different point mutations at a given position and provides links to other documents. The sentence(s) where the point mutations in KCNH2_HUMAN at position 601 were found are listed after the table.

Point mutations at position G601 in KCNH2_HUMAN

ProteinKCNH2_HUMAN (Q12809)    Gene: KCNH2,ERG, ERG1, HERG, HERG    (other point mutations)Swiss-Prot
Cross-reference table
Family page
Protein isoforms4
PositionG601
General numbering (KChannelDB) -
DomainLoop 5-6
Family alignments eag related KCNH (Kv10-12)
K+ voltage-gated channels (Kv1-12,Kca2-5)
Potassium channels 6 TMs
Other point mutations at the same position Position 601 in eag related KCNH (Kv10-12) family
Position 589 in K+ voltage-gated channels (Kv1-12,Kca2-5) family
Reference #1Laitinen P, Fodstad H, Piippo K, Swan H, Toivonen L, Viitasalo M, Kaprio J, Kontula K
Hum Mutat 2000 Jun;15(6):580-1.		Medline
Text sourceabstract
Point mutationG601S (True positive)
Reference #2Ficker E, Dennis AT, Obejero-Paz CA, Castaldo P, Taglialatela M, Brown AM
J Mol Cell Cardiol 2000 Dec;32(12):2327-37.		Medline
Text sourcePDF full text
Point mutationG601S (Not yet checked)
Reference #3Ficker E, Obejero-Paz CA, Zhao S, Brown AM
J Biol Chem 2002 Feb 15;277(7):4989-98.		Medline
Text sourceHTML full text
Point mutationG601S (Not yet checked)
Reference #4Rajamani S, Anderson CL, Anson BD, January CT
Circulation 2002 Jun 18;105(24):2830-5.		Medline
Text sourceHTML full text
Point mutationG601S (Not yet checked)
Reference #5Paulussen A, Raes A, Matthijs G, Snyders DJ, Cohen N, Aerssens J
J Biol Chem 2002 Dec 13;277(50):48610-6.		Medline
Text sourceHTML full text
Point mutationG601S (Not yet checked)
Reference #6Delisle BP, Anderson CL, Balijepalli RC, Anson BD, Kamp TJ, January CT
J Biol Chem 2003 Sep 12;278(37):35749-54. Epub 2003 Jul 1.		Medline
Text sourceHTML full text
Point mutationG601S (Not yet checked)
Reference #7Gong Q, Anderson CL, January CT, Zhou Z
Am J Physiol Heart Circ Physiol 2004 Aug;287(2):H652-8. Epub 2004 Apr 8.		Medline
Text sourceHTML full text
Point mutationS601G (Not yet checked)

Relevant sentences

Reference #1 (Laitinen P et al.): G601S
  • All these mutations are located in the evolutionarily conserved regions of HERG , including the transmembrane domains (P451L , Y569H , 1631delAG , G584S , G601S , T613M) and the cytoplasmic N-terminus (453delC , R176W) of the channel

Reference #2 (Ficker E et al.): G601S
  • For example , mature protein was not detected on Western blots of two periments , however , provided evidence that heterotetramers consisting of three WT plus one mutant hypomorphic mutations , HERG N470D and G601S , although both mutants expressed small currents at subunit left the ER and were further processed in the Golgi apparatus suggesting that at least two 37 °C.11 , 12 Recently it has been proposed that HERG A561V mutant subunits were necessary to block export

Reference #3 (Ficker E et al.): G601S
  • Here we report that the trafficking-deficient pore mutation HERG G601S was rescued by a series of HERG channel blockers that increased cell surface expression

  • In the present work we used a series of channel blockers to probe the binding site for drug-induced rescue of protein trafficking in HERG G601S

  • HERG G601S is a temperature-sensitive missense mutation in the pore region of the channel protein with most channel protein retained in the ER when expressed at physiological temperatures (18 )

  • Image densities measured in vehicle-treated (ethanol or dimethyl sulfoxide) HERG G601S controls were defined as zero protein expression levels

  • RESULTS (image)TOP (image)ABSTRACT (image)INTRODUCTION (image)EXPERIMENTAL PROCEDURES (image)RESULTS (image)DISCUSSION (image)REFERENCES Restoration of HERG G601S Trafficking by Incubation with Cisapride , E4031 , and Quinidine-- HERG G601S is located in the S5-pore helix linker and was described as a trafficking-deficient , hypomorphic LQT2 mutant expressing greatly reduced , kinetically unaltered currents when expressed in mammalian cells at physiological temperature (18 )

  • These results showed that HERG G601S is a temperature-sensitive trafficking mutant with few channels evading quality control at 37 °C

  • Restoration of HERG G601S trafficking by incubation with cisapride , E4031 , and quinidine

  • Location of mutation in HERG G601S is depicted in the extracellular part of pore region

  • Panel B1 , at 37 °C HERG G601S is synthesized only as a core-glycosylated protein of 135 kDa (con)

  • Blotted are equal amounts (20 µg) of total protein from cells expressing HERG G601S

  • Panel B2 , left lane , 135- and 155-kDa forms of HERG G601S channel protein expressed in cells treated with 5 µM cisapride at 37 °C and blotted prior to treatment with N-glycosidase F (PGNase F , -)

  • Panel C , processing of HERG G601S is shown in the presence of increasing amounts of quinidine applied in the micromolar concentration range

  • Panel D , processing of HERG G601S in the presence of increasing amounts of E4031in the micromolar concentration range

  • Panel E , concentration-dependent rescue of HERG G601S by cisapride , E4031 , and quinidine

  • We tested three HERG channel blockers for restoration of G601S trafficking at 37 °C

  • We tested whether all of these drugs restored HERG G601S trafficking in a concentration-dependent manner and whether the difference in interaction with the methanesulfonanilide binding site was reflected in their ability to rescue HERG G601S

  • HEK293 cells were transiently transfected with HERG G601S cDNA , incubated for 36 h at 37 °C , and then exposed overnight to different concentrations of cisapride (Fig. 1 B1) , quinidine (Fig. 1 C) , and E4031 (Fig. 1 D) before immunoblotting

  • The fully glycosylated 155-kDa form of the channel protein which could be detected after exposure of G601S cells to the different channel blockers was quantified using a PhosphorImager , normalized , and plotted as a function of drug concentration (Fig. 1 E)

  • All three drugs restored trafficking of HERG G601S , with half-maximal rescue concentrations (RC50) of 0.6 ± 0.1 µM for cisapride , 1.1 ± 0.2 µM for E4031 , and 1.7 ± 0.4 µM for quinidine (n = 3 or 4)

  • For WT protein both the 135- and the 155-kDa forms are synthesized at 37 °C (Fig. 2 A , con) , and expression of the mature fully glycosylated channel protein at 155 kDa is not modified significantly when cells expressing HERG WT are exposed to channel blockers at concentrations that maximally restore trafficking in HERG G601S (Fig. 2 B)

  • Channel blockers are used at concentrations that maximally restore trafficking in HERG G601S

  • To test whether the increased production of fully glycosylated mature G601S protein by HERG blockers was accompanied by increased expression of functional channels we measured G601S currents in vehicle-treated control cells and in cells exposed 48 h to saturating concentrations of cisapride (5 µM) , E4031 (5 µM) and quinidine (100 µM)

  • Fig. 3 A illustrates current recordings from a control cell expressing G601S at 37 °C and from a cell treated for 12 h with 5 µM cisapride

  • Rescued G601S channels showed no alteration in kinetic properties compared with untreated mutant channels (data not shown)

  • Despite a significant increase , current densities remained smaller in G601S than in cells transiently transfected with equal amounts of WT cDNA (382 ± 50 pA / pF)

  • Rescue of HERG G601S currents by high affinity HERG channel blockers

  • Panel A , representative whole cell current recordings from HEK293 cell expressing HERG G601S (upper part , control) and from an HEK293 cell incubated for 48 h with 5 µM cisapride (lower part of panel)

  • Panel B , G601S , current densities measured in HEK cells transfected with HERG G601S and treated post-transfection with vehicle (control) or for 48 h with 5 µM cisapride , 5 µM E4031 , 100 µM quinidine , or 5 µM astemizole

  • More than 80% of WT current levels could be recovered , indicating that the partial rescue observed in HERG G601S was not entirely the result of residual drug block

  • Panel C , normalized current inhibition measured after steady-state block with 20 nM astemizole in HERG WT and HERG G601S , HERG F656C , HERG G601S / F656C mutant channels

  • We asked whether these side chains were also important for rescue of HERG G601S

  • In immunoblotting experiments the RC50 for G601S with the parent drug astemizole was determined to be 0.06 ± 0.03 µM (Fig. 5 D)

  • HERG F656C was not trafficking-deficient but was retained in the ER when combined with the trafficking mutation 601S (HERG G601S / F656C) with trafficking being restored by incubation at 26 °C (Fig. 5 B)

  • In contrast to G601S , however , the affinity of the double mutant for astemizole block was dramatically reduced (Figs

  • Even at high concentrations only about 15% of fully glycosylated protein was recovered compared with HERG G601S

  • Panel A , Western blot analysis of equal amounts (20 µg) of total protein from cells expressing HERG G601S

  • Panel B , HERG G601S trafficking is restored by incubation at lower temperature (26 °C) ; A5.0 indicates rescue by 5 µM astemizole

  • The double mutated channel HERG G601S / F656C is rescued at a lower incubation temperature (26 °C) but not by incubation with 5 µM astemizole

  • Panel C , HERG 601S and HERG G601S / F656C are shown in the presence of increasing amounts of astemizole (A)

  • Panel D , rescue-response curves for HERG G601S incubated with increasing concentrations of astemizole and norastemizole and for HERG G601S / F656C with astemizole

  • Half-maximal rescue concentrations RC50 were 0.06 ± 0.03 µM for HERG G601S and astemizole , 1.4 ± 0.5 µM for HERG G601S and norastemizole

  • The efficacy of rescue is reduced dramatically for HERG G601S treated with norastemizole and for HERG G601S / F656C treated with astemizole (n = 3 or 4)

  • We used alkyl-TEA derivatives to probe the molecular characteristics of the rescue site in HERG G601S

  • In immunoblots we found that rescue of fully mature HERG G601S protein by these TEA derivatives was concentration-dependent (Fig. 6 , A-C)

  • A stable WT cell line was selected because it expressed much higher current densities than cells transfected with G601S and thus allowed for a more reliable determination of IC50 values

  • Moreover , we could demonstrate in experiments with astemizole that drug block was similar between HERG WT and G601S channels (Fig. 3 C)

  • Restoration of HERG G601S trafficking by quaternary ammonium compounds

  • Panel B , HERG G601S shown in the presence of increasing amounts of C8-TEA

  • Panel C , HERG G601S shown in the presence of increasing amounts of C10-TEA

  • Panel D , concentration-dependent rescue of HERG G601S by C6-TEA , C8-TEA , and C10-TEA

  • Structure-Function Studies Using Quaternary Ammonium Derivatives with Variable Headgroup Size-- To evaluate the contribution of smaller hydrophobic side chain substituents to the rescue of HERG G601S we used C8-TMA

  • Binding of both C8-TEA and C8-TMA to the channel protein was influenced by Phe-656 , and rescue by C8-TEA / TMA was strongly reduced using HERG G601S / F656C (Fig. 8 , B and C)

  • Restoration of HERG G601S trafficking by quaternary ammonium ions with reduced headgroup size

  • Panel B , HERG G601S and HERG G601S / F656C are shown in the presence of increasing concentrations of C8-TEA

  • Ast 5 indicates rescue of HERG G601S / F656C by 5 µM astemizole and was used to normalize the response

  • Panel C , concentration-dependent rescue of HERG G601S by C8-TEA and C8-TMA

  • The efficacy of rescue is reduced dramatically for HERG G601S / F656C treated with either C8-TEA or C8-TMA (n = 3 or 4)

  • We found that N-octylamine in concentrations up to 300 µM did not restore G601S trafficking

  • Restoration of HERG Trafficking with Channel Blockers Is Domain-dependent-- To this point pharmacological rescue of HERG mutants has been demonstrated for two temperature-sensitive mutations localized to the transmembrane domain: HERG G601S and HERG N470D (17 )

  • DISCUSSION (image)TOP (image)ABSTRACT (image)INTRODUCTION (image)EXPERIMENTAL PROCEDURES (image)RESULTS (image)DISCUSSION (image)REFERENCES We tested astemizole , cisapride , and E4031 , three different high affinity blockers of HERG , and the nonspecific antiarrhythmic drug quinidine for their ability to stabilize the trafficking-deficient LQT2 mutant HERG G601S in a conformation for release from the ER at physiological temperature

  • The HERG channel blockers we have used block the open state (23 , 24 , 28 ) indicating that pharmacological rescue of HERG G601S was mediated by the binding of these blockers to a receptor site in the hydrophobic inner vestibule of HERG soon after assembly of channel tetramers

  • View this table: [in this window] [in a new window] Table I Blockers of HERG WT currents as pharmacological chaperones The large differences in efficacy between drug block and rescue were not simply the result of permanent , mutation-induced structural changes in the methanesulfonanilide binding site of HERG G601S because after expression at the cell surface G601S currents were blocked to the same extent by astemizole as WT currents

  • We propose that low affinity RC50 values may be the result of small structural aberrations in the drug binding site formed early during assembly of HERG G601S channels

  • To characterize further the mechanism by which blocking molecules stabilize HERG G601S for surface expression we used alkyl-TEA derivatives thought of as generic pore blockers of potassium channels (30 , 39 )

  • Pharmacological rescue of HERG G601S followed the same rules outlined for quaternary ammonium ions as blocking molecules

  • At the same time , ligand binding stabilizes the native state of the HERG G601S channel protein

  • Based on this observation we propose that pharmacological chaperones first bind to a slightly distorted inner cavity and then reorient and stabilize the S6 helices of HERG G601S sufficiently to facilitate folding into the native , exportable state

  • Neither Mink nor MiRP1 proteins restored trafficking of HERG G601S when coexpressed (data not shown) ; however , based on our observation that rescue by pharmacological chaperones varies directly with their blocking potency it is possible that heteromultimeric HERG G601S·MirP1 mutant channels might be rescued by E4031 more effectively than HERG G601S alone

  • Up until now successful pharmacological refolding in LQT2 has only been reported for two mutations in the transmembrane domain , HERG N470D and G601S , both of which represent a mild phenotype expressing small currents at 37 °C (17 )

Reference #4 (Rajamani S et al.): G601S
  • The G601S mutation was also rescued without channel block

  • Methods (image)Top (image)Abstract (image)Introduction (image)Methods (image)Results (image)Discussion (image)References Site-Directed Mutagenesis and Transfection The LQT2 mutations N470D (asparagine to aspartic acid) , G601S (glycine to serine) , and V822M (valine to methionine) have been shown previously to be trafficking-defective channel proteins

  • 9 -11 The HERG N470D , G601S , and V822M mutations were generated by site-directed mutagenesis of wild-type HERG cDNA with the use of the GeneEditor in vitro mutagenesis system (Promega)

  • Transfection of human embryonic kidney (HEK293) cells with HERG wild-type , N470D , G601S , and V822M cDNA was carried out with Lipofectamine (Invitrogen)

  • HEK293 cells expressing the N470D , G601S , or V822M mutations were cultured at 37°C in drug-containing MEM for 24 hours

  • Pharmacological Rescue of the G601S LQT2 Mutation by Fexofenadine To test whether fexofenadine might rescue multiple HERG mutations at similar drug concentrations , we studied another trafficking-defective LQT2 mutation , G601S

  • 10 , 11 Fexofenadine-mediated pharmacological rescue of the G601S mutation is shown in Figure 3

  • The control record shows current recorded from HEK293 cells stably expressing the G601S mutation cultured at 37°C

  • The G601S-expressing cells have a small-amplitude HERG current (arrow) , indicating that some mutant channels reach the plasma membrane

  • The I-V relations for peak tail current amplitude for the G601S mutation are shown in Figure 3 B (same protocol as used in Figures 1B and 2 B)

  • Effect of fexofenadine on HERG G601S mutation

  • A small-amplitude endogenous current is present with the depolarizing steps , but unlike the N470D- and G601S-transfected cells , there is no HERG current (arrow , n = 4)

  • Our data show that the half-maximal activation voltage (V1 / 2) values for the N470D mutation are shifted negatively compared with the values obtained for wild-type or G601S mutant channels (Figures 1B , 2B , and 3 B)

  • It was recently reported by Ficker et al10 that pharmacological rescue of the G601S mutation by high-affinity HERG channel-blocking drugs varied directly with channel block potency and that rescue was disrupted by inclusion of an additional mutation within the pore-S6 drug-binding domain

  • These observations led them to propose that pharmacological rescue of the G601S mutation involved drug binding to a distorted inner vestibule in the pore-S6 region , which stabilized the protein in a configuration that improved trafficking to the plasma membrane

  • The N470D mutation is located within the S2 transmembrane-spanning domain , and the G601S mutation is located within the S5-pore extracellular linker of the HERG channel protein

  • Our data also show that the N470D and G601S mutations , but not the V822M mutation , express very-small-amplitude HERG currents when cultured under control conditions , suggesting that small numbers of channels escape the quality-control mechanism to insert into the plasma membrane

Reference #5 (Paulussen A et al.): G601S
  • For example , trafficking of mutant channels G601S , N470D , and R752W (19-21 ) was restored by lower incubation temperatures , but only G601S and N470D channels were restored by chemical chaperones

Reference #6 (Delisle BP et al.): G601S
  • Alert me when: new articles cite this article Download to Citation Manager All Versions of this Article: 278 / 37 / 35749 (most recent) M305787200v1 Thapsigargin Selectively Rescues the Trafficking Defective LQT2 Channels G601S and F805C * Brian P

  • Thapsigargin (1 µM) , a sarcoplasmic / endoplasmic reticulum Ca2+-ATPase inhibitor , rescued the surface expression of G601S , and it did so without blocking current

  • Confocal imaging showed that G601S protein is predominantly 'trapped' intracellularly and that both thapsigargin and E4031 promote its relocation to the surface membrane

  • Other sarcoplasmic / endoplasmic reticulum Ca2+-ATPase inhibitors did not rescue G601S or F805C

  • We first studied the mechanism of pharmacological rescue using the G601S LQT2 mutation , and then compared these findings with two additional LQT2 mutations , N470D and F805C

  • 18 ) , G601S (glycine to serine at position 601 ; Ref

  • Incubating HEK293 cells expressing the N470D , G601S , or F805C mutations overnight in 0.1% Me2SO had no effect on IHERG or complex glycosylation

  • Thapsigargin increases I G601S and causes complex glycosylation

  • Tail I G601S measured at -120 mV (dashed box) are shown on an expanded time base in the lower current traces

  • Incubation for 8-10 h in 1 µM thapsigargin increased I G601S

  • B , the average peak tail I G601S density for control cells (circles) and cells incubated in thapsigargin (squares) were plotted as a function of V Step and fit by Boltzmann functions (solid lines)

  • For G601S (right panel) , under control (Con) conditions only coreglycosylated protein is detected , whereas complex glycosylation of G601S appears with 5 µM E4031 or 1 µM thapsigargin exposure

  • Im munocytochemistry and Confocal Imaging—HEK293 cells expressing the G601S mutation were plated in 35-mm Petri dishes containing collagen-coated coverslips

  • RESULTS (image)TOP (image)ABSTRACT (image)INTRODUCTION (image)EXPERIMENTAL PROCEDURES (image)RESULTS (image)DISCUSSION (image)REFERENCES Incubating HEK293 Cells in Thapsigargin Increases I G601S— Thapsigargin alters cytoplasmic and ER [Ca2+] by inhibiting the sarcoplasmic / ER Ca2+-ATPase (SERCA , Ref

  • We tested if thapsigargin could also rescue the trafficking defective LQT2 mutation G601S

  • We incubated HEK293 cells expressing G601S in 1 µM thapsigargin for 8-10 h and we compared the G601S current (I G601S) to control (no drug treatment) current

  • In control conditions there is only a low amplitude tail I G601S (left panels)

  • After 8-10 h of incubation in 1 µM thapsigargin , I G601S during the depolarizing and hyperpolarizing (-120 mV) steps was increased (right panels)

  • The tail I G601S is shown in the lower panels on an expanded time base

  • Fig. 1B shows the absolute mean peak tail I G601S densities recorded at -120 mV plotted as a function of V Step (n = 5 cells in each group)

  • The data were fit with a Boltzmann function (solid line) to calculate the maximal tail I G601S density

  • Thapsigargin exposure significantly increased the maximal tail I G601S density from -11.6 ± 1.5 pA / pF in control cells to -38.2 ± 5.2 pA / pF in thapsigargin-exposed cells (p < 0.05)

  • Fig. 1C (right panel) shows a representative Western blot (n = 4) of G601S channel protein from cells incubated in 5 µM E4031 for 10 h (left lane) , control cells (middle lane) , and cells incubated in 1 µM thapsigargin for 10 h (right lane)

  • Fig. 2A shows the time course of I G601S rescue with thapsigargin after 2-3 and 8-10 h of exposure

  • Cells expressing G601S were depolarized for3sto20mVto fully activate I G601S and tail currents were recorded at -120 mV

  • Representative tail I G601S from control cells and from cells exposed to 1 µM thapsigargin show a progressive , time-dependent increase in the tail I G601S amplitude

  • Fig. 2B shows the absolute mean peak tail I G601S density from control cells and cells incubated in thapsigargin

  • After incubating 2-3 h , thapsigargin increased I G601S density (n = 5 cells in each group , p < 0.05) , which was further increased after 8-10 h

  • I G601S did not increase further after 8-10 h of exposure

  • Incubation in thapsigargin for 2-3 h rescues I G601S and complex glycosylation

  • A , tail I G601S was measured by using a holding potential of -80 mV , applying a depolarizing step to 20 mV for 3 s , followed by a hyperpolarizing step to -120 mV for 3 s , and repeated every 20 s

  • Representative tail currents recorded during the hyperpolarizing step are shown for control (Con) cells , and I G601S was increased in cells incubated in 1 µM thapsigargin for 2-3 , 8-10 , or 24 h

  • B , thapsigargin increased the absolute mean peak tail I G601S density in a time-dependent manner compared with control (Con) cells (* , p < 0.05)

  • Thapsigargin Does Not Block I G601S—We wanted to determine whether thapsigargin rescue of G601S protein involved drug block of the channel

  • We first pharmacologically rescued I G601S by incubating G601S expressing cells overnight with 5 µM E4031

  • From the holding potential of -80 mV , I G601S was recorded by depolarizing cells to 0 mV for 3 s , then repolarizing to -50 mV for 5 s to record tail current

  • The voltage protocol was repeated every 20 s , and I G601S was recorded before and during the application of 1 µM thapsigargin or 5 µM E4031

  • Fig. 3A shows representative current traces of pharmacologically rescued I G601S before and during the application of 1 µM thapsigargin (upper panel) or 5 µM E4031 (lower panel)

  • Thapsigargin , unlike E4031 , did not cause block of I G601S

  • Fig. 3B shows averaged normalized peak tail I G601S amplitude data recorded at -50 mV from G601S expressing cells , and the effect of thapsigargin or E4031

  • Thapsigargin did not affect the peak tail I G601S (circles)

  • In contrast , 5 µM E4031 rapidly blocked I G601S (triangles)

  • We conclude that thapsigargin-induced pharmacological rescue of G601S does not involve drug block of I HERG

  • Thapsigargin does not cause block of I G601S

  • I G601S was rescued by incubating cells overnight in 5 µM E4031 , and E4031 was then removed

  • From the same cells , the records show control (Con) I G601S and I G601S after the application of 1 µM thapsigargin (+Thapsi , 4 min) or 5 µM E4031 (+E4031 , 1 min) to the bath solution

  • Thapsigargin did not block I G601S , whereas E4031 blocked I G601S leaving a small amplitude endogenous current native to HEK293 cells

  • B , the averaged normalized peak tail I G601S amplitude is plotted as a function of time before and after the application of 1 µM thapsigargin or 5 µM E4031 (drug applied at 0 time)

  • I G601S did not change after the application of thapsigargin (circles)

  • In contrast , E4031 rapidly blocked I G601S (triangles)

  • Pharmacological Rescue of G601S Protein Requires the Golgi Apparatus—Thapsigargin and E4031 rescue I G601S and cause complex glycosylation of the protein

  • This suggests that pharmacological rescue by both drugs results in G601S trafficking through the Golgi apparatus where complex sugars are modified and added to the protein

  • We tested the hypothesis that disruption of the Golgi apparatus with BFA would prevent pharmacological rescue of G601S

  • For this experiment , G601S expressing cells were incubated with 1 µM thapsigargin or 5 µM E4031 for 2-3 h to induce pharmacological rescue of the channel

  • As shown in Fig. 2 , this incubation period is sufficient to rescue G601S channels

  • Fig. 4A shows representative tail I G601S recorded at -120 mV from cells cultured with and without BFA , and Fig. 4B shows the absolute mean peak tail I G601S density (n = 4 cell in each group)

  • BFA reduced I G601S rescue in cells exposed to thapsigargin or E4031 (p < 0.05 in each group)

  • Because co-incubating BFA inhibited the increase of I G601S by either thapsigargin or E4031 exposure , the data suggest that pharmacological rescue is dependent on an intact Golgi apparatus

  • A , representative tail I G601S recorded using the same voltage protocol as Fig. 2 are shown after 2-3 h exposure to thapsigargin (left traces) or E4031 (right traces)

  • Co-incubating the cells with 5 µg / µl BFA prevented the pharmacological rescue of I G601S

  • B , the absolute mean peak tail I G601S was smaller in cells co-incubated in BFA compared with cells incubated in only thapsigargin or E4031 (* , p < 0.05)

  • Pharmacological Rescue Redistributes Intracellular G601S Protein to the Cell Surface—The results suggest that incubation in thapsigargin or E4031 mobilizes G601S protein that is present in an intracellular compartment(s)

  • To test this , we used confocal microscopy to image G601S protein in control cells , and in cells incubated for 10 h with 1 µM thapsigargin or 5 µM E4031 to induce pharmacological rescue

  • The phase-contrast images show the cell and its surface membrane , the z-scan images show G601S HERG protein staining

  • The single and stacked z-scan images show that G601S HERG protein staining is predominantly perinuclear in control cells with only faint staining into the cell processes and cell surface membrane

  • Thapsigargin causes the redistribution of G601S protein from perinuclear compartments to the cell surface

  • The localization of G601S protein in cells was imaged using im munocytochemistry and confocal microscopy

  • The left column pictures show phase-contrast images of G601S-expressing HEK293 cells

  • For control conditions (row Con) G601S protein is predominantly perinuclear with little antibody staining on the cell membrane surface

  • Incubating cells in 1 µM thapsigargin (row Thapsi) or 5 µM E4031 (row E4031) for 10 h increased G601S staining at the surface membrane , shown in both the single and stacked z-scan images

  • Thapsigargin and E4031 Selectively Rescue Different LQT2 Mutations—Three methods to increase functional I G601S are reduced culture temperature (<30 °C , Ref

  • We compared findings obtained with the G601S mutation with two other trafficking defective LQT2 mutations

  • Fig. 6A shows representative tail currents recorded at -120 mV from cells stably expressing the G601S , F805C , or N470D mutations under control conditions ; after incubating at 27 °C for 24 h ; and after incubating in thapsigargin (1 µM) or E4031 (5 µM) for 24 h

  • In contrast , E4031 selectively increased peak tail I G601S and I N470D densities , but not I F805C density

  • Incubating cells in thapsigargin for 24 h selectively increased peak tail I G601S and I F805C densities , but not I N470D density (n = 5-8 cells in each group)

  • Complex glycosylation was apparent only in N470D and G601S after incubation in E4031 , and only in G601S and F805C after incubation in thapsigargin

  • A , representative tail I G601S , I F805C , and I N470D recorded using the same voltage protocol as in Fig. 2 from control cells (Con) , cells incubated at 27 °C for 24 h , cell incubated in 5 µM E4031 for 24 h , or cells incubated in 1 µM thapsigargin (Thapsi) for 24 h

  • The N470D mutation alters the deactivation rate of the channel , causing I N470D to decay slower than I G601S or I F805C

  • B , absolute mean peak tail current densities of I G601S , I F805C , and I N470D recorded at -120 mV from control cells , cells incubated at 27 °C for 24 h , cells incubated in E4031 for 24 h , or cells incubated in thapsigargin for 24 h

  • Compared with control , incubating cells at 27 °C increased the mean peak tail I G601S , I F805C , and I N470D ; incubating cells in E4031 increased only I G601S and I N470D ; incubating cells in thapsigargin increased only I G601S and I F805C (* , p < 0.05)

  • C , representative Western blots (n = 4 for each group) show that the appearance of the 155-kDa protein band correlates with the increases in current densities shown in B for G601S , F805C , or N470D

  • Other SERCA Inhibitors Do Not Rescue G601S or F805C—We tested whether a different SERCA inhibitor could also rescue G601S or F805C by using DBHQ (10 µM)

  • In control cells (n = 5) and for cells incubated with 10 µM DBHQ (n = 4) for 8-10 h the mean peak tail I G601S density remained unchanged , 8 ± 2 pA / pF

  • Similarly , incubating G601S expressing cells in DBHQ (10 µM) or cyclopiazonic acid (1 or 10 µM) , another inhibitor of the SERCA , for 10 h failed to cause complex glycosylation of G601S protein (data not shown , n = 5 and 2 , respectively)

  • 4-PBA also rescues {Delta}F508 CFTR after 48 h of exposure (14 ) , therefore we tested if 4-PBA could increase G601S trafficking

  • We conclude that unlike thapsigargin , 4-PBA does not rescue trafficking of the G601S mutation , and this is in agreement with the previous report that 4-PBA did not rescue I N470D (9 )

  • DISCUSSION (image)TOP (image)ABSTRACT (image)INTRODUCTION (image)EXPERIMENTAL PROCEDURES (image)RESULTS (image)DISCUSSION (image)REFERENCES We show that thapsigargin causes pharmacological rescue of the LQT2 mutations G601S and F805C

  • 9 ) was shown to rescue I G601S and I N470D at drug concentrations below that required for block of I HERG

  • The Mechanism of Thapsigargin-induced Rescue of G601S Is Different Than {Delta}F508 CFTR—The mechanism proposed for thapsigargin-induced pharmacological rescue of the {Delta}F508 CFTR mutation was SERCA inhibition , and finding that other SERCA inhibitors such as DBHQ and cyclopiazonic acid also rescued the trafficking of {Delta}F508 CFTR supported this (12 )

  • The principal difference is that other SERCA inhibitors failed to rescue the G601S or F805C mutation

  • Thus one possibility is that SERCA inhibition similar to that found with the {Delta}F508 CFTR mutation is not responsible for the rescue of G601S

  • Our findings do show that thapsigargin-induced rescue of G601S was evident within 2-3 h of drug exposure

  • Pharmacological Rescue Requires the Golgi Apparatus for Surface Expression of G601S—Complex glycosylation , or the generation of the 155-kDa protein band on Western blot , is a hallmark of HERG protein maturation and pharmacological rescue (2 , 5 , 6 )

  • The im munocytochemical and confocal imaging results support this ; pharmacological rescue redistributed G601S protein from predominantly perinuclear intracellular compartment(s) to the cytoplasm and surface membrane

  • Furthermore , disrupting the Golgi apparatus using BFA prevented rescue of I G601S

  • We conclude that the G601S mutation is trapped within an intracellular compartment , presumably the ER (1 , 6 ) , and that pharmacological rescue of the G601S channel protein with both thapsigargin and E4031 requires an intact Golgi apparatus for complex glycosylation and subsequent transport into the surface membrane

  • In summary , these findings show that thapsigargin selectively rescues the G601S and F805C , but not the N470D LQT2 mutant channels

  • Furthermore , given the differences in the pharmacological rescue of N470D , G601S , and F805C , we suggest that trafficking of these mutations is differentially regulated and that multiple rescue mechanisms may exist

  • 1 The abbreviations used are: HERG , human ether-a-go-go-related gene ; LQT , long QT ; ER , endoplasmic reticulum ; I HERG , HERG current ; CFTR , cystic fibrosis transmembrane conductance regulator ; 4-PBA , 4-phenylbutyrate ; SERCA , sarcoplasmic / endoplasmic Ca2+-ATPase ; IG601S , G601S current ; BFA , brefeldin A ; I F805C , F805C current ; I N470D , N470D current ; DBHQ , 2 , 5-(di-tert)-butyl-1 , 4-hydroquinone

Reference #7 (Gong Q et al.): S601G
  • We and other investigators have shown that the trafficking defective LQT2 mutations T65P , N470D , and S601G can be rescued by HERG channel blocking drugs (5 , 20 , 21 , 29 )

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F.Horn (kchanneldbcmbi.ru.nl), 17-Aug-2005