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

Ying Ke,Chai Ann Ng,Matthew D. Perry,Jamie I. Vandenberg,Mark J. Hunter

doi:10.1074/jbc.m113.531277

Ying Ke, Chai Ann Ng + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m113.531277

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Abstract

The N-terminal cytoplasmic region of the Kv11.1a potassium channel contains a Per-Arnt-Sim (PAS) domain that is essential for the unique slow deactivation gating kinetics of the channel. The PAS domain has also been implicated in the assembly and stabilization of the assembled tetrameric channel, with many clinical mutants in the PAS domain resulting in reduced stability of the domain and reduced trafficking. Here, we use quantitative Western blotting to show that the PAS domain is not required for normal channel trafficking nor for subunit-subunit interactions, and it is not necessary for stabilizing assembled channels. However, when the PAS domain is present, the N-Cap amphipathic helix must also be present for channels to traffic to the cell membrane. Serine scan mutagenesis of the N-Cap amphipathic helix identified Leu-15, Ile-18, and Ile-19 as residues critical for the stabilization of full-length proteins when the PAS domain is present. Furthermore, mutant cycle analysis experiments support recent crystallography studies, indicating that the hydrophobic face of the N-Cap amphipathic helix interacts with a surface-exposed hydrophobic patch on the core of the PAS domain to stabilize the structure of this critical gating domain. Our data demonstrate that the N-Cap amphipathic helix is critical for channel stability and trafficking.

Highlights

A cytoplasmic PAS domain regulates Kv11.1 function, but its role in channel assembly is unclear
PAS Domain Is Not Required for Trafficking, Assembly, or Stability of Kv11.1 Channels—Transfection of HEK293 cells with full-length Kv11.1a gives rise to two bands on Western blot, one at ϳ135 kDa, which represents the core-glycosylated protein, and another at ϳ155 kDa, which represents the fully glycosylated (FG) protein (Fig. 1B)
The other serine mutants had a normal trafficking phenotype (Fig. 7B). These results suggest that the hydrophobic face of the N-Cap amphipathic helix is important for normal trafficking of Kv11.1a channels

Summary

Introduction

A cytoplasmic PAS domain regulates Kv11.1 function, but its role in channel assembly is unclear. Results: PAS domain deletion does not alter assembly, but removal of the N-Cap that immediately precedes the PAS domain severely disrupts channel trafficking. Conclusion: The N-Cap is vital for PAS domain stability and channel trafficking. When the PAS domain is present, the N-Cap amphipathic helix must be present for channels to traffic to the cell membrane. Serine scan mutagenesis of the N-Cap amphipathic helix identified Leu-15, Ile-18, and Ile-19 as residues critical for the stabilization of full-length proteins when the PAS domain is present. Mutant cycle analysis experiments support recent crystallography studies, indicating that the hydrophobic face of the N-Cap amphipathic helix interacts with a surface-exposed hydrophobic patch on the core of the PAS domain to stabilize the structure of this critical gating domain. Our data demonstrate that the N-Cap amphipathic helix is critical for channel stability and trafficking

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