The present experiments demonstrate that both wild type hERG and the N470D mutant associated with calnexin. The association of hERG channels with calnexin requires N-linked glycosylation. Recently, it has been reported that more than one glycan is needed for efficient trimming of glucose by glucosidase II and as a consequence, many glycoproteins with a single glycan fail to associate with calnexin (30
). We have previously shown that although hERG contains two consensus N-linked glycosylation sites N598 and N629, only N598 is used for glycosylation (20
). The fact that newly synthesized hERG proteins associate with calnexin suggests that a single glycan is sufficient to mediate the interaction with calnexin. Several other glycoproteins with a single glycan, especially membrane proteins such as erythrocyte anion exchanger AE1, V2 vasopressin receptor, and Kv1.2 potassium channel, have been reported to associate with calnexin (25
). One possibility is that many of these membrane proteins are known to form dimers or tetramers, and activation of glucosidase II may occur during oligomerization (14
The observation that only the immature, but not the mature form of hERG associates with calnexin is consistent with the function of calnexin as an ER chaperone. Calnexin is a lectin chaperone protein that interacts with glycan moieties of the glycoproteins. Calnexin plays an important role in the quality control of glycoproteins in a process termed calnexin cycle. In this process, calnexin associates with glycoproteins that are monoglucosylated intermediates of the N-linked core glycan. The association of calnexin with monoglucosylated glycoproteins is regulated by a cycle of deglucosylation, by glucosidase II, and reglucosylation, by UDP-glucose:glycoprotein glucosyltransferase (UGGT). Since UGGT preferentially acts on unfolded proteins, only the incompletely folded proteins reenter the cycle, while the properly folded proteins leave the ER and move further along the secretory pathway. Thus, our results suggest that the wild type hERG channel transiently associates with calnexin during the early stages of biogenesis, and dissociates from calnexin when it folds properly. In contrast, the N470D mutant has a prolonged association with calnexin, suggesting that N470D fails to fold properly, is recognized by UGGT, and reenters the calnexin cycle.
The interaction of calnexin with voltage-gated potassium channels has been reported in Shaker and Kv1.2 channels (33
). For Shaker channels, calnexin is not involved in the quality control and ER retention of mutant Shaker channels (35
). However, transient calnexin interaction confers long-term stability of folded Shaker channel proteins in the ER and promotes surface expression of correctly assembled Shaker channels (36
). Similarly, calnexin facilitates cell surface expression of Kv1.2 potassium channels (33
In order to study the folding of hERG channels, we performed trypsin digestion experiments. The results show that the immature and mature forms of wild type hERG were different in their sensitivity to digestion by trypsin. The core-glycosylated hERG was about 100-fold more sensitive to trypsin digestion than the complex-glycosylated mature form. This suggests that the trypsin-sensitive sites are more readily accessible in the core-glycosylated immature form but are probably hidden in the mature form as a result of proper folding. Our results also show that in the presence of BFA, a fraction of the 135 kDa form of wild type hERG is in a folding conformation that is comparable to the 155 kDa mature form even though its trafficking to the Golgi is blocked by BFA. This fraction may represent the properly folded hERG protein that would have trafficked to the cell surface in the absence of BFA. This result suggests that the conformation change from the loosely folded form to the proper folded form occurs in the ER.
Defective protein trafficking has been recognized as an important mechanism for an increasing number of inherited human diseases (38
). In many cases, trafficking defective mutant proteins are functional if they can be rescued to their final destinations. Recently, the use of specific ligands as pharmacological chaperones has emerged as a strategy for rescue of trafficking defective proteins (39
). It has been hypothesized that binding of specific ligands to the unfolded or misfolded proteins promotes correct folding. We and other investigators found that the trafficking defective LQT2 mutations R28E, T65P, N470D and G601S can be rescued by hERG channel blockers (21
). It has been shown that pharmacological rescue requires binding of the drugs to the inner vestibule of the pore region of the hERG channel (41
Our present results show that in the presence of E-4031, the N470D mutant is able to escape the calnexin cycle and exit the ER. Therefore, E-4031 binding may improve proper folding of the mutant channel so that it is no longer a substrate for UGGT. The protease sensitivity experiments show that the complex-glycosylated mature form of the N470D mutant protein rescued by E-4031 becomes more resistant to trypsin digestion, suggesting that it has a compact conformation that is similar to the mature form of wild type hERG. In addition, the results demonstrate that pharmacological rescue of the N470D mutant by E-4031 is associated with its increased resistance to trypsin even in the conditions that its trafficking from the ER to the Golgi is blocked by BFA. This indicates that the E-4031 induced conformational changes take place in the ER.
LQT2 mutations that can be rescued by hERG channel blocking agents appear to express small amplitude currents under control conditions 21, 41-43). This observation is consistent with our trypsin sensitivity findings that the conformation of N470D is similar to wild type folding intermediates in the ER, as both show a similar sensitivity to trypsin. In addition, the fact that both wild type hERG and N470D were completely soluble in 0.1% Triton X-100 in the detergent extraction experiments suggests that the N470D mutant does not cause significant aggregation of the hERG channel protein. Our previous pulse-chase experiments showed that the immature form of wild type hERG can be efficiently converted to the mature form, whereas the immature form of N470D cannot (9
). This observation indicated that the immature form of wild type hERG is in an incompletely folded intermediate state, which can become the properly folded mature form and traffic to the plasma membrane. However, the presence of the mutation in N470D may result in the formation of a hemodynamic hurdle that inhibits maturation of the mutant channel and consequently causes its ER retention. Taken together, the results from the protease digestion and detergent extraction studies, and previous pulse-chase experiments suggest that the N470D mutant is not grossly misfolded but is trapped as partially folded intermediates that are structurally similar to the immature form of wild type protein. Similar findings have been reported for the CFTR ΔF508 and mutant P-glycoproteins (26
). Thus, the LQT2 mutations that can be rescued by hERG channel blockers may represent a mild phenotype with subtle folding defects and a low efficiency of maturation, and hERG channel blocking agents may act as pharmacological chaperones to increase the maturation efficiency of these mutant channels by promoting correct folding of channel proteins.
The use of pharmacological chaperones to rescue trafficking defective mutant proteins has been shown in a variety of human diseases (39
). In most cases, however, the mechanisms of pharmacological rescue are not fully understood. Our present findings provide evidence that hERG channel blocker E-4031 restores proper folding of trafficking defective mutant channels and promotes their cell surface expression. Although pharmacological rescue of trafficking defective mutant channels has potential implications as a therapeutic approach for LQT2 patients, the hERG channel blocking agents such as E-4031 are not suitable for this purpose. However, elucidating the mechanisms by which E-4031 rescues hERG mutant channels will facilitate the search for new pharmacological chaperones that can restore trafficking of mutant channels without channel blocking properties.