Changes in Channel Trafficking and Protein Stability Caused by LQT2 Mutations in the PAS Domain of the HERG Channel

Abstract

Inherited human long-QT2 syndrome (LQTS) results from mutations in the gene encoding the HERG channel. Several LQT2-associated mutations have been mapped to the amino terminal cytoplasmic Per-Arnt-Sim (PAS) domain of the HERG1a channel subunit. Here we have characterized the trafficking properties of some LQT2-associated PAS domain mutants and analyzed rescue of the trafficking mutants by low temperature (27°C) or by the pore blocker drug E4031. We show that the LQT2-associated mutations in the PAS domain of the HERG channel display molecular properties that are distinct from the properties of LQT2-associated mutations in the trans-membrane region. Unlike the latter, many of the tested PAS domain LQT2-associated mutations do not result in trafficking deficiency of the channel. Moreover, the majority of the PAS domain mutations that cause trafficking deficiencies are not rescued by a pore blocking drug. We have also explored the in vitro folding stability properties of isolated mutant PAS domain proteins using a thermal unfolding fluorescence assay and a chemical unfolding assay.

A TRPV4 Channel C-terminal Folding Recognition Domain Critical for Trafficking and Function

“…We propose that mutational effects are probably results of disruption of the conformation of this region required for the normal protein maturation process. Rescue of TRPV4 G849A/P851A channel function by lowering incubation temperature is fully consistent with this hypothesis, indicating that misfolding is probably the cause for ER retention and loss of function, as previously documented in other misfolding proteins (30,48,49). Given that segment deletions within a broad neighboring region of the C terminus were tolerable, results from the present study are best explained by a working model assuming that the region around residues of Gly 849 and Pro 851 is essential for correct folding of the TRPV4 channel.…”

“…Functional Rescue of TRPV4 G849A/P851A Mutant by Lowering Temperature-To test the hypothesis that the lack of complex glycosylation and dysfunction of the deletion mutants were caused by protein misfolding, we tested whether lowering temperature could rescue mutant channel function (48,49). HEK293 cells transfected with either TRPV4dm or TRPV4dmG849A/P851A were incubated at 30°C for 24 -48 h before Western blot analysis.…”

Role of the Cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) Domain in Trafficking and Stabilization of Kv11.1 Channels

“…The Kv11.1a PAS domain is a hot spot for clinical mutations that result in reduced stability of the isolated PAS domain and decreased trafficking of the full-length channel (17,19). This is consistent with the hypothesis that the PAS domain plays an important role in channel assembly and/or stability of channels once they reach the plasma membrane.…”

“…Thus, in Kv11.1-3.1, which has a partially truncated PAS domain, the trafficking is poor. Second, LQT2 mutants that alter stability of the PAS domain result in improper folding and cause impaired trafficking (17,19). The data in this study clearly demonstrate that if the PAS domain is present, then the N-Cap amphipathic helix of the PAS domain must also be present (Fig.…”

“…How then can we explain the numerous studies in the literature indicating that mutations in the PAS domain affect trafficking of full-length Kv11.1a channels (17,19,31) and in many instances affect domain-domain interactions involving the PAS domain (17,18)? First, if the PAS domain is present, then it must be fully intact and properly folded.…”