| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
Cellular dysfunction of LQT5-minK mutants: abnormalities of IKs, IKr and trafficking in long QT syndrome
Introduction
Results
Clinical phenotypes
Electrophysiological effects of wild-type and mutant minKs on KvLQT1 and HERG currents
Cellular processing of wild-type and mutant minKs
Discussion
Materials And Methods
Mutation analysis
Mutagenesis and cRNA preparation
Oocyte preparation and injection
Cell culture and transfection procedures
Electrophysiology
Immunocytochemistry
Acknowledgements
References
Cellular dysfunction of LQT5-minK mutants: abnormalities of IKs, IKr and trafficking in long QT syndrome
Received March 10, 1999; Revised and Accepted May 11, 1999
Mutations in the minK gene KCNE1 have been linked to the LQT5 variant of human long QT syndrome. MinK assembles with KvLQT1 to produce the slow delayed rectifier K+ current IKs and may assemble with HERG to modulate the rapid delayed rectifier IKr. We used electrophysiological and immunocytochemical methods to compare the cellular phenotypes of wild-type minK and four LQT5 mutants co-expressed with KvLQT1 in Xenopus oocytes and HERG in HEK293 cells. We found that three mutants, V47F, W87R and D76N, were expressed at the cell surface, while one mutant, L51H, was not. Co-expression of V47F and W87R with KvLQT1 produced IKs currents having altered gating and reduced amplitudes compared with WT-minK, co-expression with L51H produced KvLQT1 current rather than IKs and co-expression with D76N suppressed KvLQT1 current. V47F increased HERG current but to a lesser extent than WT-minK, while L51H and W87R had no effect and D76N suppressed HERG current markedly. Thus, V47F interacts with both KvLQT1 and HERG, W87R interacts functionally with KvLQT1 but not with HERG, D76N suppresses both KvLQT1 and HERG, and L51H is processed improperly and interacts with neither channel. We conclude that minK is a co-factor in the expression of both IKs and IKr and propose that clinical manifestations of LQT5 may be complicated by differing effects of minK mutations on KvLQT1 and HERG.
INTRODUCTION
The long QT syndrome (LQTS) is characterized by a prolonged QT interval,due to delayed repolarization of the cardiac action potential. Although the clinical phenotype is variable even within the same family, affected individuals are more prone to syncope,torsade de pointes and ventricular fibrillation, sometimes leading to sudden death (1).
LQTS may be autosomal dominant [Romano-Ward syndrome (RW) (2)] or autosomal recessive, expressing also deafness [Jerve and Lange-Nielsen syndrome (JLN) (3)]. LQTS is a heterogeneous gene disorder and has been linked to four ion channel genes: KCNQ1 categorized as LQT1 (4); KCNH2, LQT2 (5); SCN5A, LQT3 (6); and KCNE1, LQT5 (7,8). LQT4 has been mapped to human chromosome 4q25-27 but its gene has not been identified. The genes linked to LQTs 1, 2 and 5 encode cardiac K+ channels that produce the slowly activating delayed rectifier K+ current IKs and the rapidly activating delayed rectifier K+ current IKr (9). IKr can be reproduced by expression of HERG, the product of KCNH2 (10,11), while IKs can be reproduced by co-expression of KvLQT1 (KCNQ1) and minK or Isk, the product of KCNE1. MinK is a regulatory subunit of KvLQT1 and is essential for reproduction of an appropriately gated IKs of enhanced amplitude (12,13). MinK may also contribute to the ion conduction pathway (14-17). However, minK may not be restricted to KvLQT1 in its K+ channel interactions; it has also been shown to affect HERG amplitude and gating (18), raising the possibility that both IKs and IKr currents are compromised in LQT5.
Two of five identified LQT5 mutations [T7I, T59P, L60P, S74L and D76N (7,8,19)] have been studied functionally up to now [S74L and D76N (19,20)] and have been shown to reduce I19,20Ks and alter gating in Xenopus oocytes. The possible effects on IKr have not been compared although, in CHO cells, mutation D77N (the rat counterpart of the human D76N) was co-immunoprecipitated with HERG without changing amplitude or gating (18). In the present study, we compared the effects of three different missense LQT5 mutations, discovered recently in two families (21), and the D76N mutation (20,22) on the expression of IKs and IKr currents. Because missense mutations associated with LQT2 were processed imperfectly (23), we tagged wild-type and mutated minKs to follow trafficking to the cell surface.
RESULTS
Clinical phenotypes
Mutations V47F and L51H were identified in a 20-year-old woman with a prolonged QT interval (QTc 520 ms) and a bilateral hearing deficit. The proband inherited mutation V47F from her mother and L51H from her father. Both parents have a normal QT interval and normal hearing function. She remains asymptomatic without treatment.
A 14-year-old girl carrying mutation W87R was brought to our attention after prolongation (QTc 500 ms) of the QT interval was discovered during a normal check up. She has no cardiac history and remains asymptomatic. Four other family members were found to be carriers of the same mutations and are asymptomatic despite prolongation of the QT interval (Fig. 1).
Figure 1. Pedigree structures of LQTS families. Black symbols denote carriers of the minK mutations.
Electrophysiological effects of wild-type and mutant minKs on KvLQT1 and HERG currents
To determine their functional consequences, we co-expressed wild-type and mutant minKs with KvLQT1 and with HERG in Xenopus oocytes and HEK293 cells, respectively. Equimolar ratios of KvLQT1 and mutant minK cRNAs were co-injected into oocytes and the resulting currents were compared with currents produced by co-expression of KvLQT1 and WT-minK. HEK293 cells were chosen to study the effect of minK mutations on HERG in order to minimize contamination from the IKs current that would occur due to the presence of endogenous KvLQT1 channels in oocytes (13). A HERG/HEK cell line was developed, and wild-type and mutant minKs were transiently transfected as described in Materials and Methods.
Figure 2 shows the effects of WT-minK on KvLQT1 and HERG currents. In oocytes, WT-minK greatly increased the KvLQT1 current level, delayed its activation and shifted its voltage dependence to more positive potentials (12,13,24). The effect of WT-minK on HERG expressed in HEK cells consisted of a doubling of current amplitude and a shift of the activation curve by about -8 mV (Fig. 2B) (18). In mammalian cells, HERG activation time constants were found to be of the order of seconds between -40 and 0 mV (11,25); therefore, we determined the voltage dependence of HERG by measuring tail currents at -120 mV after 30 s depolarizations (Fig. 2B). With shorter (2 s) voltage steps, the midpoint of the activation curve was shifted from -22 to -6 mV. IKs currents at room temperature do not attain a steady-state level even after very long depolarization pulses; therefore, their voltage dependence was determined empirically by constructing isochronal (t = 2.7 s) activation curves.
Figure 2. Effects of WT-minK on KvLQT1 and HERG. (A) Examples of currents expressed in Xenopus oocytes, following injection with KvLQT1 cRNA (250 ng/µl) minus (left panel) and plus minK cRNA (50 ng/µl, middle panel). Currents were elicited by depolarizing voltage steps from -60 and -40 mV to +40 and +60 mV, respectively, from a holding potential of -80 mV, in 20 mV increments. The return potential was -50 mV. Right panel: the normalized activation curve obtained from five oocytes injected with KvLQT1 (open squares) and the normalized isochronal (t = 2.7 s) activation curve from 10 oocytes injected with KvLQT1 + minK (filled squares). The KvLQT1 activation curve was obtained with voltage steps from -60 to +50 mV in 10 mV increments. Experimental data points were fitted with the equation 1/[1 + exp(V - V1/2)/k], which gave the following V1/2 and slope factors: V1/2 = -26.7 mV, slope = 14.3 mV for KvLQT1; and V1/2 = 9.3 mV, slope = 16.8 mV for KvLQT1 + minK. (B) Examples of currents in HEK293 cells stably transfected with HERG (left panel) and following transient transfection with minK (middle panel). Currents were generated by depolarizing steps in 10 mV increments from -60 to 0 mV from a holding potential of -80 mV. The return potential was -120 mV. Right panel: activation curves obtained from HERG- (open squares) and HERG + minK-expressing cells (filled squares). Experimental data points were best fit with V1/2 = -22.1 mV, slope = 7.9 mV for HERG; and V1/2 = -30.5 mV, slope = 7.4 mV for HERG + minK. n = 3-5 for each data point. Values are means ± SE.
All four minK mutations (Fig. 3A) produced abnormal IKs and IKr currents compared with WT-minK. Both KvLQT1-IKs and HERG-IKr were decreased in each case (Fig. 3B and Table 1), but by varying amounts. At equivalent minK concentrations and a constant concentration of KvLQT1, V47F produced 50-70% of the amplitude increase produced by WT-minK,and a similar result was observed for HERG-IKr. Neither L51H nor W87R produced any increase in either KvLQT1-IKs or HERG-IKr. Mutation D76N suppressed both currents below the control levels observed in the absence of WT-minK (Fig. 3B). Previous results showed that rat minK mutant D77N, corresponding to human D76N, failed to change HERG currents (18). Therefore, we also tested the effect of D76N on HERG-IKr expressed in Xenopus oocytes. Co-expression of D76N with HERG (250 ng/µl) produced currents that were smaller than HERG currents alone. The effect was dose dependent,with up to nearly 100% suppression using a D76N cRNA concentration of 500 ng/µl (Fig. 3C). To test whether the current suppression by D76N was specific for KvLQT1 and HERG, we expressed it with Kv 4.3. D76N failed to affect the Kv 4.3 current (Fig. 3D). We also tested the effect of WT-minK and L51H on HERG in Xenopus oocytes. In order to minimize contamination by minK co-assembling with endogenous KvLQT1 in HERG-IKr current measurements, high concentrations of HERG cRNA and short voltage protocols were used. Nevertheless, that small portion of current corresponding to minK co-assembling with endogenous KvLQT1 was subtracted in our analysis. Wild-type and L51H minKs had the same effects on HERG in oocytes as were observed in HEK cells (Fig. 3D).
Figure 3. KvLQT1 and HERG current enhancement by wild-type and mutant minKs. (A) The mutations are shown in relation to the predicted topology of minK. The c-Myc epitope fused to the N-terminus of the protein is indicated by the gray box. Branched structures indicate potential glycosylation sites. e, extracellular; i, intracellular. (B) The effects of wild-type and mutant minKs on KvLQT1 and HERG current levels. Oocytes were co-injected with KvLQT1 (250 ng/µl) and minK (50 ng/µl); for the injection with both V47F and L51H, concentrations were 25 ng/µl for each cRNA. IKs currents were measured at +40 mV (t = 2.7 s) from a holding potential of -80 mV and compared with the currents from oocytes injected with KvQLT1 alone. n = 6-10. The dotted line indicates the KvLQT1 current level. Right panel: HEK293-HERG cells were transiently transfected either with wild-type or mutant minKs. HERG tail currents were measured at -120 mV after 30 s depolarizations at 0 mV and normalized by the current obtained in HERG-expressing cells. All currents were normalized by the cell capacitance. The dotted line indicates the HERG current level in stably transfected cells. n = 5-10. (C) Effect of mutant D76N on HERG expressed in Xenopus oocytes. Current-voltage relationships are shown for HERG (250 ng/ml, squares, n = 11) and HERG + D76N (100 ng/ml, circles, n = 7; and 500 ng/ml, triangles, n = 6). Currents were elicited by 2 s voltage steps from -60 to +40 mV from a holding potential of -80 mV, in 20 mV increments. (D) Comparison of the effect of D76N on HERG and Kv 4.3 current amplitudes and the effect of wild-type and L51H on HERG in Xenopus oocytes. Current ratios are currents produced in minK + HERG or + Kv 4.3 and the currents obtained with HERG or Kv 4.3 alone. For D76N, the current ratio is obtained from the value at 0 mV shown in (C). Oocytes were co-injected with Kv 4.3 (100 ng/µl) + D76N (500 ng/µl) or HERG (125 ng/µl) + wild-type, D76N or L51H (100 ng/µl). HERG currents were measured at 0 mV from a holding potential of -80 mV. Kv 4.3 currents were measured at +80 mV, the holding potential was -80 mV. For WT-minK + HERG current measurements, the voltage step at 0 mV was 0.5 instead of 2 s. Values were compared with HERG alone produced by the same length voltage step. Prior to normalization, that portion of current corresponding to minK co-assembling with endogenous KvLQT1 was subtracted from that of WT-minK + HERG. n = 11, 6, 7 and 9, respectively. All values are means ± SE.
Table 1. Ability of wild-type and mutant minKs to enhance KvLQT1 and HERG currents
| KvLQT1 | HERG | HERG | |
| in oocytes | in HEK293 | in oocytes | |
| Wild-type | 9.8 ± 1.5 | 1 | 1.2 ± 0.2 |
| (n = 6) | (n = 2) | ||
| V47F | 6.8 ± 2.9 | 0.61 ± 0.13 | NA |
| (n = 6) | (n = 2) | ||
| L51H | -0.26 ± 0.14 | 0.065 ± 0.01 | 0.03 |
| (n = 3) | (n = 2) | ||
| ½ V47F + ½ L51H | 6.3 ± 2.5 | NA | NA |
| (n = 4) | |||
| D76N | -1 | -0.51 ± 0.06 | -0.67 ± 0.01 |
| (n = 2) | (n = 2) | ||
| W87R | 0.4 | -0.08 ± 0.04 | NA |
| (n = 2) |
The proband having V47F is a compound heterozygote with mutation L51H on the other minK allele (21). We tested the effects of both mutations by co-injecting them together with KvLQT1 in oocytes. The currents were not significantly different from those obtained with co-assembly of V47F and KvLQT1 (Fig. 213B and Table 1).
In order to compare the biophysical properties of wild-type and mutant minKs, we characterized their effects on the voltage dependence and kinetics of deactivation of KvLQT1-IKs and HERG-IKr (Fig. 4 and Table 2, respectively). Currents generated by co-assembly of KvLQT1 and V47F were half-maximal at +38.8 mV, a 22.2 mV positive shift compared with WT-IKs currents (Fig. 4B). The kinetics of deactivation were also faster ([tau] = 0.73 0.05 s versus 1 0.04 s for WT at -50 mV; Table 2), confirming a change in the channel gating. On the other hand, the effect of V47F on HERG voltage dependence was similar to that of WT-minK,with the half-maximum activations for WT and V47F at -30.5 and -29.1 mV, respectively. The deactivation kinetics were also similar. No change in deactivation kinetics was observed for WT-minK or any of the minK mutations expressed with HERG (Table 2).
Figure 4. Voltage dependence of currents expressed with minK mutants. (A) Currents of KvLQT1 + minK mutants, HERG and HERG + minK mutant were produced by the same voltage protocols as described in Figure 1. (B) Normalized isochronal (t = 2.7 s) activation curves obtained from oocytes co-injected with KvLQT1 and wild-type or mutant minK. Experimental data points were fitted with the equation 1/[1 + exp(V - V1/2)/k], which gave the following V1/2 and slope factors: V1/2 = +16.6 mV, slope = 22.4 mV for KvLQT1 + minK (filled squares, n = 9); V1/2 = +38.8 mV, slope = 18.7 mV for KvLQT1 + V47F (open circles, n = 7); V1/2 = -4.5 mV, slope = 20 mV for KvLQT1 + L51H (open squares, n = 5); V1/2 = +38 mV, slope = 19.9 mV for KvLQT1 + V47F + L51H (open triangles, n = 10); and V1/2 = +48.8 mV, slope = 21.9 mV for KvLQT1 + W87R (open diamonds, n = 8). (C) Activation curves obtained from HEK cells expressing HERG + wild-type or mutant minK. Experimental data points were best fitted with: V1/2 = -30.5 mV, slope = 7.5 mV for HERG + minK (filled squares); V1/2 = -29.1 mV, slope = 8.5 mV for HERG + V47F (open circles); V1/2 = -26.5 mV, slope = 8.4 mV for HERG + L51H (open squares); V1/2 = -21.3 mV, slope = 8 mV for HERG + D76N (open triangles); and V1/2 = -26.1 mV, slope = 5.6 mV for HERG + W87R (open diamonds). n = 5-8. Values are mean ± SE.
Table 2. Deactivation time constants of minK + KvLQT1 and minK + HERG
| KvLQT1 (s) | HERG (ms) | ||
| [tau]1 | [tau]2 | ||
| Wild-type | 1 ± 0.04 | 28.4 ± 2.6 | 158.6 ± 11.8 |
| (n = 8) | (n = 8) | (n = 8) | |
| V47F | 0.73 ± 0.05** | 32.3 ± 2.5 | 164.7 ± 16.9 |
| (n = 7) | (n = 4) | (n = 4) | |
| L51H | ND | 28.7 ± 3.7 | 156.4 ± 8.9 |
| (n = 5) | (n = 5) | ||
| ½ V47F + ½ L51H | 0.83 ± 0.03* | NA | NA |
| (n = 9) | |||
| D76N | ND | 27.5 ± 1.9 | 139.7 ± 14.6 |
| (n = 4) | (n = 4) | ||
| W87R | 0.24 ± 0.009** | 35.2 ± 2.3 | 159.3 ± 26.9 |
| (n = 7) | (n = 6) | (n = 6) |
Mutation W87R shifted the midpoint of the KvLQT1-IKs activation curve to +48.7 mV and increased the rate of closure of the channel ([tau] = 0.24 0.009 s at -50 mV). For HERG, there was a steeper voltage dependence (k = 5.4 versus 7.5 mV for WT-minK) and a smaller shift (~5 versus 8 mV for WT-minK) of the activation curve to more negative potentials (Fig. 4C). As noted, D76N suppressed KvLQT1 and HERG currents. Analysis of the remaining HERG current revealed that it had the same characteristics as the current in the stably transfected HEK cells, with a midpoint for the activation curve of -21.3mV and a slope factor of 8 mV (Fig. 4C).
KvLQT1 (Fig. 3B, left panel) and HERG (Fig. 3B, right panel, and D) currents were not changed by L51H. Currents recorded from L51H + KvLQT1 (Fig. 4A) were similar to currents produced by KvLQT1 alone (Fig. 2A). The voltage dependence was not identical to that of homomultimeric channels, with a midpoint value of -4.5 mV (Fig. 4B) versus -26.7 mV (Fig. 2A). L51H co-expressed with HERG produced currents with an activation curve shifted to -26.5 versus -22 mV. Co-expression of V47F + L51H with KvLQT1 gave currents with a voltage dependence indistinguishable from that of V47F + KvLQT1.
MinK sites were proposed to line the pore of IKs (14-17). We tested whether minK mutations might change selectivity by measuring the current reversal potentials in solutions containing 5 mM K+ and 100 mM Na+. The current reversal potential was approximately -80 mV for wild-type, V47F and L51H, and approximately -70 mV for W87R (Fig. 5).
Figure 5. K+ selectivity of wild-type and mutant IKs currents. Currents were generated by the voltage protocol shown in the inset and normalized to the maximal current. Currents were elicited in a solution containing 5 mM KCl and 100 mM NaCl. The reversal potentials were: -78 mV for KvLQT1 + minK (filled squares, n = 4); -78 mV for KvLQT1 + L51H (open circles, n = 5); -82 mV for KvLQT1 + V47F (open triangles, n = 5); and -70 mV for KvLQT1 + W87R (open diamonds, n = 4).
Cellular processing of wild-type and mutant minKs
Our electrophysiological results suggested that mutants V47F and W87R were transported to the cell surface together with KvLQT1 to regulate the amplitudes and gating of IKs. Other possibilities existed for L51H and D76N, such as retention of mutant minK, or minK mutant transported to the cell surface but non-functional. L51H failed to change either IKs or IKr. In Xenopus oocytes, currents produced by injection of L51H + KvLQT1 appeared similar to those produced by KvLQT1 alone. D76N suppressed both currents but it was not clear whether this phenomenon was occurring at the plasma membrane or whether it was the result of intracellular retention of KvLQT1 and HERG subunits by D76N.
We tested for the presence of wild-type and minK mutations at the plasma membrane using tagged constructs with c-Myc fused to the putative extracellular N-terminus. As a control, the c-Myc-WT-minK was co-expressed with KvLQT1 in Xenopus oocytes. The currents were indistinguishable from currents produced by WT-minK + KvLQT1, suggesting that the epitope did not interfere with normal processing or function (data not shown). To ensure that incubation with the primary antibody occurred in conditions that preserved plasmalemmal integrity, we incubated cells with the c-Myc antibody in complete media and at 37°C in humidified air containing 5% CO2, and we fixed them only after extensive washing of the antibody from the cell surface.
Figure 6 shows examples of immunofluorescence staining of WT-minK. Transient transfection of tagged WT-minK in HEK293 cells resulted in expression of the protein in ~10% of the cells (Fig. 6A and B). Unpermeabilized c-Myc-WT-minK cells showed a clear ring of stain around the surface of the cell when focusing through the cell diameter, and patches of stain when focusing at the cell's upper extremity (Fig. 6C and D). Similar patterns were observed for c-Myc-D76N and c-Myc-W87R (Fig. 7), suggesting that these mutations likewise are transported to the cell surface. In contrast, c-Myc-L51H-transfected cells did not display any fluorescence immunostaining. To determine if L51H was retained intracellularly, experiments were repeated using permeabilized cells. These cells displayed clusters of stain around the nucleus (Fig. 7). Permeabilized WT-minK-transfected cells retained a pattern of staining that was similar to that of non-permeabilized cells but with more patchy staining when focusing through the cell diameter, probably as a result of immunodetection of proteins trafficking from locations such as the endoplasmic reticulum to the plasma membrane (data not shown).
Figure 6. Immunolocalization of WT-minK at the cell surface. The c-Myc epitope fused to the N-terminus of the protein was used for immunolocalization of minK. Transiently transfected cells were incubated with anti-c-Myc antibody in complete media at 37°C prior to fixation. Cells were then blocked with 5% non-fat dry milk in PBS plus 0.1% Tween-20 and incubated with rhodamine-conjugated sheep anti-mouse antibody. (A) Phase contrast light transmission image of HEK cells transiently transfected with WT-minK. (B) Immunofluorescence image of the same field shown in (A). (C) Immunofluorescent image of a cell transfected with c-Myc-WT-minK. (D) Different focus of the same image shown in (C). Images were acquired with an Olympus inverted microscope equipped with a Spot32 digital camera and software from Diagnostic Instruments.
Figure 7. Immunofluorescence staining of minK mutants. Immunofluorescence images of c-Myc-D76N, c-Myc-W87R and c-Myc-L51H. For D76N and W87R, cells were incubated with the anti-c-Myc antibody prior to fixation. For c-Myc-L51H, a large field (note the calibration bar) is shown from an experiment conducted similarly, indicating lack of fluorescence staining at the cell surface. A c-Myc-L51H-transfected cell is also shown from an experiment in which cells were fixed and permeabilized with Triton X-100 (0.1%), prior to incubation with anti-c-Myc antibody (L51H-triton).
DISCUSSION
Each of the four LQT5 missense mutations we studied had a unique cellular phenotype. This result predicts variability in the clinical phenotype, which has in fact been reported (7,8,20,21). One of the mutations, L51H, was not processed to the cell membrane. This mutation had no effect on HERG current probably because there was no interaction with the HERG channel. The typical IKs current produced by WT-minK was not observed; rather the current was typical for that expressed by KvLQT1 alone. However, the mutant produced a shift in the voltage dependence of a typical KvLQT1 current. Two explanations may be invoked: first, overexpression of a non-functional protein might activate endogenous currents with similar properties (26); second, cytoplasmically retained L51H protein might affect KvLQT1 currents. Some support for this possibility may be the observation that small peptides corresponding to the cytoplasmic domain of minK were able to produce IKs-like currents when injected into Xenopus oocytes (27). In any event, the effect is thought to be minor.
The three remaining mutations were expressed at the cellular surface. D76N clearly interacted strongly with both KvLQT1 and HERG to suppress the currents which they express. Discordant results have been obtained by different groups on the functional effect of D76N minK on KvLQT1 channels (19,20). One group detected complete suppression of KvLQT1 by D76N (20), as we did, and a strong dominant-negative effect of the mutant on WT-IKs. The other group (19) observed functional IKs channels following co-expression of D76N with KvLQT1, although their properties were distinct from those of WT-IKs. The same group previously reported lack of expression and a dominant-negative effect of the rat D77N on the current produced in Xenopus oocytes by co-assembly of minK with endogenous KvLQT1 (28). Their explanation for their own discordant results invoked the use of Xenopus versus human KvLQT1 to produce IKs currents. This does not apply to our case since we used human KvLQT1 as they did. It was also reported that a D77N-HERG complex was immunoprecipitated from plasma membrane fractions of CHO cells but, unlike the present experiments, there was no change in HERG current (18). Under our experimental conditions, we found that D76N suppressed KvLQT1 and HERG currents in both HEK293 cells and Xenopus oocytes. We have no ready explanation for this difference, but both sets of results support the hypothesis that minK is a modulator of HERG as well as of KvLQT1.
Both V47F and W87R interacted with KvLQT1 to produce IKs-like currents having voltage dependences and amplitudes clearly different from those manifested by WT-minK + KvLQT1. We interpret the small shift of the reversal potential of W87R to be due to leak contamination that is more pronounced in oocytes expressing small currents. KvLQT1 + L51H, although equally small, may not show the same shift because the current expressed is KvLQT1 alone, which was shown to have a higher K+/Na+ permeability ratio (29). This predicts a slightly more negative current reversal potential, which could be canceled by the summation with leak current and brought back to a value close to KvLQT1 + minK (-78 mV). However, we cannot exclude the possibility that the shift indicates a true change in selectivity introduced by W87R. Between V47F and W87R, only V47F affected HERG currents. W87R, like L51H, produced no effect on HERG current but, unlike L51H, was expressed at the cell surface. We cannot exclude the possibility that W87R assembles with HERG but that it does not have functional effects on the channel.
The proband who was a compound heterozygote had both mutations, L51H and V47F. Co-expression of the two mutants gave rise to currents similar in amplitude and voltage dependence to V47F + KvLQT1 currents. We consider this additional evidence that L51H produces unprocessed minK subunits. These results also indicate that L51H does not interfere with the interaction between V47F and KvLQT1.
The compound heterozygote displays a mild JLN phenotype, with only partial hearing deficit and less severe cardiac phenotype. We suggest that the milder clinical phenotype is predicted by the nature of the cellular phenotypes. The partial function of V47F and the lack of interference by L51H with other minK subunits may ensure normal cardiac function in heterozygotes. W87R produces, on the other hand, a more severe destruction of IKs function. All carriers of a single mutant allele in fact present prolongation of the QT interval. D76N presents the most severe clinical phenotype, with complete hearing loss, cases of sudden death and frequent syncope episodes (7,8). Our results indeed show that D76N is the mutation causing the most severe loss of repolarizing K+ current.
To summarize, each of the four LQT5 mutations we have studied expressed a cellular phenotype that was clearly distinguishable from WT-minK. The results taken together also provide strong evidence for the hypothesis that minK may assemble with HERG as well as with KvLQT1. Unlike LQTs1-3, LQT5 involves not one but two cardiac ion channels and, as a result, is expected to produce a more complex clinical phenotype.
MATERIALS AND METHODS
Mutation analysis
DNA was extracted from peripheral blood lymphocytes using standard procedures (30). Single-strand conformation polymorphism (SSCP) analysis was performed on amplified genomic DNA encoding the minK gene, obtained by PCR using specific primers. Abnormal conformers (not present in 200 controls) were sequenced manually as described previously (31).
Mutagenesis and cRNA preparation
Mutations were prepared by PCR overlap extension (32) using human minK cDNA as template. Flanking oligonucleotides were synthesized to introduce a HindIII restriction site and the Kozak consensus sequence upstream of the initiating ATG codon, and a BamHI restriction site following the stop codon. In addition, primers containing the mutations were prepared. PCR products were subcloned into the pCR 2.1 vector (Invitrogen, Carlsbad, CA) for sequence verification and amplification. Plasmids containing the mutations were digested with HindIII and BamHI, and the resulting 390 bp fragments were subcloned into pSP64 (Promega, Madison, WI) and pCDNA3 (Invitrogen) vectors. For immunological detection of the minK mutants, a c-Myc epitope (EQKLISEEDL) was fused to the N-terminus of the wild-type protein by PCR. pCDNA3 vectors containing the minK mutants (with and without the c-Myc tag) were used for transient transfection of HEK293 cells. MinK mutants subcloned in pSP64 were used for in vitro cRNA synthesis. pSP64 plasmids were linearized with EcoRI and cRNA was prepared with the mMESSAGE mMACHINE kit (Ambion, Austin, TX) using SP6 RNA polymerase. cRNAs were dissolved in 0.1 M KCl and their size and integrity were evaluated by formaldehyde-agarose gel electrophoresis. Concentrations were estimated by comparison with a 0.24-9.5 kb RNA ladder (Gibco BRL, Gaithersburg, MD). All cRNAs were diluted to the final desired concentration in 0.1 M KCl and injected into oocytes.
Oocyte preparation and injection
Xenopus oocytes were surgically removed and enzymatically defolliculated using collagenase (2 mg/ml, 1.5 h) in calcium-free OR2 solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2 and 5 mM HEPES, pH 7.6). Stage V-VI oocytes were injected with 46 nl of cRNA and incubated at 19°C in SOS solution (100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES and 2.5 mM pyruvic acid, pH 7.6) + gentamycin (100 µg/ml).
Cell culture and transfection procedures
The human embryonic kidney cell line (HEK293) was used for expression of the minK constructs. Cells were cultured in minimal essential medium (MEM; Gibco BRL) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL), 100 U/ml penicillin and 100 µg/ml streptomycin (complete media) at 37°C in a humidified atmosphere containing 5% CO2. HEK293 cells were stably transfected with HERG cDNA cloned in pCNA3 using Lipofectin as DNA carrier and geneticin as selection marker. The construct was digested with SalI prior to transfection, to facilitate incorporation into the HEK chromosomal DNA. Geneticin (0.5 mg/ml)-resistant clones were amplified and the expression of HERG confirmed by PCR and by electrophysiological experiments.
Lipofectin was also used for transient transfections. Cells were plated the day before transfection at 70-80% confluence on plates or coverslips. DNA at equivalent concentrations for wild-type and minK mutants was pre-complexed with lipids in MEM without FBS and without antibiotics at room temperature for 45 min following the recommended DNA:lipids ratios. Transfection was performed at 37°C in a humidified atmosphere containing 5% CO2 and was stopped after 4 h by substituting the transfection media with complete media. Cells were cultured for an additional 24-48 h prior to use. At least two separate transfections were performed for all of the minKs examined and, on average, eight cells per transfected batch were studied functionally. For electrophysiological experiments, transfected cells were identified using the green fluorescent protein (pEGFP-C2; Clontech, Palo Alto, CA) or the lymphocyte surface antigen CD8-[alpha] ([pi]H3-CD8), whose plasmids were added at a ratio of 0.5:1 to the minK DNA. Magnetic polystyrene beads coated with antibody to CD8 (Dylan, Oslo, Norway) were added to the cells 5-10 min prior to the experiments.
Electrophysiology
Voltage-clamp of Xenopus oocytes.
Currents were recorded at room temperature 3-5 days after injection using the conventional double-electrode technique with the commercially available amplifier OC-725B (Warner Instruments, Hamden, CT). Pipettes were filled with 3 M KCl and had resistances of 1-2 M[Omega]. Oocytes were perfused with a solution containing 100 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 2 mM MgCl2 and 10 mM HEPES (pH adjusted to 7.4 with NaOH). Current densities were not calculated since the experiments used stage V-VI oocytes, which had comparable sizes. In any given batch of oocytes, currents produced by mutant + KvLQT1 were always compared with currents obtained from WT-minK + KvLQT1. The voltage dependence of activation was determined empirically at isochronal times of 2.7 s.
Patch-clamp of HEK293 cells.
The whole-cell configuration of the patch-clamp method was applied. Currents were recorded at room temperature with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA), using pipettes that had resistances of 2-5 M[Omega]. Cells were perfused with a solution containing 137 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 1.5 mM MgCl2, 10 mM HEPES and 10 mM glucose (pH adjusted to 7.3 with NaOH), and pipettes were filled with a solution containing: 140 mM K-aspartate, 1 mM MgCl2, 10 mM EGTA and 10 mM HEPES (pH adjusted to 7.3 with KOH). Currents were normalized by the capacitance of the cell. Capacitive currents were evoked by -5 mV voltage step from a holding potential VH of -60 mV and compensated by the analog circuit of the amplifier. The readout of the amplifier correction was used to estimate cell capacitance. Since minK was transfected transiently, transfected cells were recognized either as fluorescent or as binding beads coated with CD8 antibodies, and untransfected cells in the same preparation were used as controls.
Data acquisition and analysis were performed with the pClamp suite of programs. Data were filtered at 0.2 kHz and digitized at 0.7 kHz for IKs and at 1 and 5 kHz for IKr current measurements, respectively.
Immunocytochemistry
Transiently transfected cells were incubated in fresh complete media containing the monoclonal anti-c-Myc antibody (40 µg/ml, clone 9E10; Boehringer Mannheim, Indianapolis, IN) at 37°C for 1 h. Cells were then washed once with phosphate-buffered saline (PBS) and fixed with paraformaldehyde (4% in PBS) for 15 min at room temperature. After fixing, cells were washed three times for 5 min with PBS and blocked for 1 h at room temperature with 5% non-fat dry milk in PBS plus 0.1% Tween-20. Cells were then incubated with the secondary antibody, rhodamine-conjugated sheep anti-mouse (1:50, in 5% non-fat dry milk in PBS plus 0.1% Tween-20; Cappel, Organon Teknika, Boxtel, The Netherlands), for 1 h at room temperature and subsequently washed three times for 5 min with PBS. For the experiments with permeabilized cells, cells were washed once with PBS, fixed with paraformaldehyde, washed three times for 5 min with PBS and incubated with Triton X-100 (0.1% in PBS) for 5 min, prior to incubation with the blocking solution (5% non-fat dry milk in PBS + 0.1% Tween-20). The incubation with the primary antibody was performed at 37°C for 1 h or at room temperature for 2 h. The glass coverslips were mounted with VECTOREX medium (Vector, Burlingame, CA). Cells were examined with an Olympus inverted microscope equipped with a Spot32 digital camera from Diagnostic Instruments (Sterling Heights, MI), using a 40× or a 100× objective lens. Images were acquired with the software supplied by Diagnostic Instruments and, analyzed and mounted with Adobe Photoshop 5.0 for Windows 95.
ACKNOWLEDGEMENTS
We thank Dr W.-Q. Dong and C.-D. Zuo for oocytes handling and injection, Dr Barbara Wible for the HERG/HEK293 cell line, and Dr Krystyna Surewicz for helpful comments. This work was supported by the National Institutes of Health (NIH grants NS23877, HL36930 and HL55404 to A.M.B.), by the American Heart Association (AHA grant 9804566 to L.B.) and by the Italian Telethon Foundation (TIGEM, grants 748 and 1058 to S.G.P., C.N. and P.J.S.).
REFERENCES
*Present address: Department of Pharmacology, Vanderbilt University Medical Center, 23rd Avenue South at Pierce, Nashville, TN 37232, USA
+To whom correspondence should be addressed. Tel: +1 216 778 5960; Fax: +1 216 749 3889; Email: abrown{at}research.mhmc.org
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