The C-terminal HRET sequence of Kv1.3 regulates gating rather than targeting of Kv1.3 to the plasma membrane

András Balajthy,Gyorgy Panyi,Péter Hajdu,Sándor Somodi,Orsolya Voros,Orsolya Szilagyi

doi:10.1038/s41598-018-24159-8

Abstract

Kv1.3 channels are expressed in several cell types including immune cells, such as T lymphocytes. The targeting of Kv1.3 to the plasma membrane is essential for T cell clonal expansion and assumed to be guided by the C-terminus of the channel. Using two point mutants of Kv1.3 with remarkably different features compared to the wild-type Kv1.3 (A413V and H399K having fast inactivation kinetics and tetraethylammonium-insensitivity, respectively) we showed that both Kv1.3 channel variants target to the membrane when the C-terminus was truncated right after the conserved HRET sequence and produce currents identical to those with a full-length C-terminus. The truncation before the HRET sequence (NOHRET channels) resulted in reduced membrane-targeting but non-functional phenotypes. NOHRET channels did not display gating currents, and coexpression with wild-type Kv1.3 did not rescue the NOHRET-A413V phenotype, no heteromeric current was observed. Interestingly, mutants of wild-type Kv1.3 lacking HRET(E) (deletion) or substituted with five alanines for the HRET(E) motif expressed current indistinguishable from the wild-type. These results demonstrate that the C-terminal region of Kv1.3 immediately proximal to the S6 helix is required for the activation gating and conduction, whereas the presence of the distal region of the C-terminus is not exclusively required for trafficking of Kv1.3 to the plasma membrane.

Highlights

Potassium channels are essential players in setting the membrane potential and in the regulation of intracellular signaling in both excitable and non-excitable cells[1,2]
We introduced the A413V mutation in the S6, which has been shown to accelerate dramatically the inactivation kinetics of A413V homomers as compared to wild-type channels. As it was shown earlier, heterotetramers consisting of A413V and the wild type (WT) subunits display intermediate inactivation kinetics between τA413V and τWT depending on the number of mutant subunits in the tetrameric channel[22,24,25]
We found that a CHO cell expressing both A413V-NOHRET and WT-Kv1.3 exhibits currents resembling the pure WT current (Fig. 5A); and not the “mixture” of multiple heteromeric currents characterized with various inactivation time constants

Summary

Introduction

Potassium channels are essential players in setting the membrane potential and in the regulation of intracellular signaling in both excitable and non-excitable cells[1,2]. Lu et al showed that Shaker K+ channels, a Kv1 analogue in Drosophila, are targeted to the plasma membrane without the “HRE” region of the C-terminal. Deletion of amino acids preceding the “HRET” sequence in Shaker, (still part of the C-terminal region) prevented channel expression. Kv1.3 was shown to be guided to the cell membrane by the presence of two acidic glutamate (E) residues at the C-terminus of the channel: removal of these amino acids with C-terminal deletion or mutation to isoleucine resulted in the loss of ionic current and plasma membrane expression[14]. Truncation of the C-terminal including this motif may interfere with the activation gate and results in cell-surface targeting but a non-conducting construct. The objective of this study was to clarify whether the removal of the major part of the C-terminal tail of Kv1.3 – with or without the HRET(E)-region – has any impact on the cell-surface trafficking or/and channel conductance of Kv1.3

Objectives

Methods

Results

Conclusion