KChannelDB: Extraction of mutation data from the literature

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

The table below describes the selected point mutation and provides links to other documents. The sentence(s) where the point mutation R752W was found are listed after the table.

The mutated residues are also indicated in the family sequence alignments and hyperlinked to the corresponding mutation pages.

Point mutation R752W in KCNH2_HUMAN

Relevant sentences:

R752W

• We studied a novel COOH-terminal missense mutation , HERG R752W , which segregated with the disease in a family of 101 genotyped individuals

• Therefore , HERG R752W represents a new class of trafficking mutants in hLQTS

• In a large LQT2 family we found a novel COOH-terminal missense mutation , HERG R752W , which was retained in the ER

• When trafficking was corrected , HERG R752W channels conducted enhanced currents that slightly shortened the cardiac action potential simulated by the Luo-Rudy model of a mammalian ventricular myocyte

• HERG R752W was prepared by overlap extension PCR , verified by sequencing , and subcloned as a Xho I-BamH I fragment into either full-length HERG-pSP64 or HERG-pCDNA3

• For the purpose of this study , the formulation of the fast delayed rectifier potassium current , I Kr , was modified to fit our experimental voltage clamp data obtained from HERG-WT and HERG-R752W channels

• RESULTS (image)TOP (image)ABSTRACT (image)INTRODUCTION (image)MATERIALS AND METHODS (image)RESULTS (image)DISCUSSION (image)REFERENCES HERG R752W was identified in a 31-yr-old woman with a history of syncope who died suddenly

• Characterization of HERG R752W currents in Xenopus oocytes

• After injection of equivalent amounts of cRNA , we were surprised to find that mutant HERG R752W channels produced larger outward currents than WT channels (Fig. 1 , A and B)

• Both WT and R752W produced typical bell-shaped isochronal current-voltage (I-V) relationships (Fig. 1 C)

• For R752W , the amplitudes were larger and the I-V curve was shifted to more hyperpolarized potentials

• Compared with the corrected WT , I-V curve threshold and peak for R752W remained shifted (Fig. 1 C) , suggesting that altered gating properties contributed importantly to the larger currents of R752W

• Coinjection of equal amounts of WT and R752W cRNA produced a summation of current amplitudes at potentials more positive than -20 mV

• At more hyperpolarized potentials , current amplitudes from the combined injection were not significantly changed from R752W alone despite the increased amount of cRNA injected

• One explanation might be that below -20 mV , activation rates for R752W / WT heteromultimers are very different from homomultimeric channels (see Fig. 2 C) , thereby producing a complex interaction with current amplitudes as measured in Fig. 1 C

• HERG R752W is located in the COOH-terminal linker between transmembrane domain S6 and the cyclic-nucleotide binding fold common to all members of the ether-a-go-go gene family with the arginine at this position being highly conserved

• Macroscopic current recordings from Xenopus oocytes injected with 0.5 ng each of HERG wild-type (WT ; A) or HERG R752W cRNA (B ; RW)

• D: maximal tail current amplitudes elicited on return to -90 mV after a test pulse to 60 mV were back-extrapolated and used to assess the average number of functional channels expressed in oocytes injected with WT or R752W cRNA

• Half-maximal inactivation V k was -63.3 ± 1.1 mV for WT and -55.5 ± 1.6 mV for HERG R752W (n = 4)

• HERG R752W accelerates current activation (A)

• HERG R752W speeds channel deactivation (D)

• triangle , Time constants measured on coinjection of equal amounts of WT and R752W cRNA

• To analyze the gating changes introduced by R752W , we studied steady-state activation and inactivation (Fig. 1 , E and F)

• Compared with WT , the activation and inactivation curves of R752W were shifted by +11.9 and +7.8 mV , respectively (Fig. 1 F)

• R752W channels activated much faster than WT channels , and this acceleration contributed to increased outward currents with shorter depolarizations

• Activation time constants were intermediate between WT and R752W at -20 and 0 mV but approximated values measured for R752W alone at +20 and +40 mV (Fig. 2 C)

• Coinjections of equal amounts of WT and R752W produced intermediate values for slow time constants

• For the faster process , coinjections approached values measured for R752W alone at potentials between -90 and -120 mV

• On the other hand , the faster activation rates of R752W predicted more current during the plateau and a tendency to shorten the cardiac action potential

• Simulations of the effects of WT HERG R752W current on the cardiac action potential

• Therefore , we expressed HERG R752W in mammalian HEK293 cells at a physiological temperature of 37°C and used whole cell patch clamp recordings to characterize mutant currents

• We were not able to detect any HERG-like outward current in HEK293 cells transfected with R752W , although WT current was obtained routinely under identical experimental conditions (Fig. 4 , A and B)

• Because mutant currents were elicited in Xenopus oocytes and because oocytes are maintained at lower temperatures than mammalian cells , we tested the effect of reduced incubation temperature on expression of R752W

• After incubation for 2 days at 26°C , large whole cell HERG currents were recorded in cells transfected with R752W cDNA and had an electrophysiological profile similar to that described in Xenopus oocytes (Fig. 4 , C and D)

• 3.8 mV for HERG WT) reflects faster current activation of HERG R752W

• To evaluate effects of incubation temperature on current expression , HEK293 cells were transfected with either 2 µg HERG WT or 2 µg HERG R752W cDNA and incubated for 2 days at 26 or 37°C as indicated

• In coexpression experiments , HEK cells were transfected with either 1 µg HERG WT + 1 µg vector cDNA (coWT37) or 1 µg HERG WT + 1 µg HERG R752W (WT / RW37) cDNA and incubated for 2 days at 37°C

• These results suggested that R752W was not processed correctly at 37°C and processing resumed when the incubation temperature of mammalian cells was lowered to 26°C

• In contrast , HERG R752W channel protein synthesized at 37°C was present only in the core-glycosylated 135-kDa form and a somewhat smaller form representing either an unglycosylated form (18 ) or more likely a degradation product (Fig. 5 C)

• Accordingly , immunostaining of R752W-transfected cells with anti-HERG antibodies produced strong perinuclear staining consistent with the accumulation of protein in the ER

• When cells transfected with R752W were incubated at 26°C , we detected the 160-kDa band corresponding to the fully mature protein as well as the immature bands

• The same bands were observed in Western blots of crude membrane fractions from Xenopus oocytes injected with either WT or R752W cRNA

• Because defects in processing of the temperature-sensitive Delta F508 CFTR protein were partially corrected by chemical chaperones such as glycerol (4 , 24 ) or transcriptional regulators such as 4-phenylbutyrate (4-PB) (20 ) , we tested whether these agents might correct the malfunction of R752W processing

• In none of these experiments did we observe any change in the glycosylation pattern of R752W incubated at 37°C , whereas protein synthesis was progressively decreased with higher glycerol concentrations (Fig. 5 C)

• Patch clamp recordings also failed to detect R752W currents in cells incubated for 2 days at 37°C at the different glycerol concentrations

• Furthermore , treatment of R752W transfected cells with 2.5 mM 4-PBA at 37°C had no effect on processing and no currents were detectable although protein synthesis was substantially increased (Fig. 5 C)

• In contrast , HERG R752W channel protein synthesized at 37°C was present only in the core-glycosylated form when transfected cells were treated for 24 h in the presence of 5 µM E-4031 (Fig. 5 C)

• DISCUSSION (image)TOP (image)ABSTRACT (image)INTRODUCTION (image)MATERIALS AND METHODS (image)RESULTS (image)DISCUSSION (image)REFERENCES The electrophysiological phenotype of the processed R752W mutant is characterized by mild changes in gating properties that do not translate into prolongation of the cardiac action potential and hence the QT interval as judged by our simulations

• The accelerated activation and deactivation kinetics of R752W resembles mutations described previously in the NH2-terminal PAS (Per , Arnt , and Sim) domain of the channel protein , especially HERG R56Q (5 )

• At reduced temperatures where protein folding might be altered , HERG R752W attained a conformation that ultimately permitted exit from the ER

• We explored whether chemical chaperones known to stabilize temperature-sensitive mutants were effective in correcting the trafficking defect associated with HERG R752W

• In CFTR it was shown that glycerol promoted the maturation of mutants K464R and K464A but not of K464W , a mutation with an amino acid substitution similar to the one analyzed in HERG R752W

• The failure of glycerol treatment to facilitate trafficking of HERG R752W was surprising because trafficking of HERG N470D , a hypomorphic mutation in the transmembrane domain of HERG , was improved by incubation in 10% glycerol (31 )

• Similarly , the antiarrhythmic drug E-4031 facilitated trafficking of HERG N470D , but was ineffective on the R752W mutant

• These differences suggest that HERG R752W represents a new and different class of misprocessed mutants with distinct mechanisms responsible for retention of mutations localized to different functional domains of the HERG channel protein

• Because chemical chaperones were not effective in HERG R752W , we complemented our experiments by treating HEK cells expressing HERG R752W with 4-PB , an analog of the transcriptional regulator butyrate

• In analogy to experiments in CFTR , we tried to boost expression of HERG R752W to force the mutant by mass action past the ER to the plasma membrane (20 )

• Although synthesis of HERG R752W was substantially enhanced , we were not able to detect fully glycosylated mature protein

• In summary , HERG R752W is the first member of a novel class of temperature-sensitive trafficking mutants that do not express any current at 37°C and are resistant to rescue by osmolytes or specific channel ligands

• Thus , although HERG R752W behaves as a loss-of-function mutation , there is one major difference from hLQT mutations reported heretofore: HERG R752W encodes a fully functional channel protein if it can be made to reach the plasma membrane

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