Decreased Subunit Stability as a Novel Mechanism for Potassium Current Impairment by a KCNQ2 C Terminus Mutation Causing Benign Familial Neonatal Convulsions

Vincenzo Barrese,Pasqualina Castaldo,Giulia Bellini,Maurizio Taglialatela,Antonio Pascotto,Stefano Bonatti,Francesco Miceli,Maria Virginia Soldovieri,Lucio Annunziato,Luisa Iodice,Emanuele Miraglia del Giudice

doi:10.1074/jbc.m510980200

Abstract

KCNQ2 and KCNQ3 K+ channel subunits underlie the muscarinic-regulated K+ current (I(KM)), a widespread regulator of neuronal excitability. Mutations in KCNQ2- or KCNQ3-encoding genes cause benign familiar neonatal convulsions (BFNCs), a rare autosomal-dominant idiopathic epilepsy of the newborn. In the present study, we have investigated, by means of electrophysiological, biochemical, and immunocytochemical techniques in transiently transfected cells, the consequences prompted by a BFNC-causing 1-bp deletion (2043deltaT) in the KCNQ2 gene; this frameshift mutation caused the substitution of the last 163 amino acids of the KCNQ2 C terminus and the extension of the subunit by additional 56 residues. The 2043deltaT mutation abolished voltage-gated K+ currents produced upon homomeric expression of KCNQ2 subunits, dramatically reduced the steady-state cellular levels of KCNQ2 subunits, and prevented their delivery to the plasma membrane. Metabolic labeling experiments revealed that mutant KCNQ2 subunits underwent faster degradation; 10-h treatment with the proteasomal inhibitor MG132 (20 microm) at least partially reversed such enhanced degradation. Co-expression with KCNQ3 subunits reduced the degradation rate of mutant KCNQ2 subunits and led to their expression on the plasma membrane. Finally, co-expression of KCNQ2 2043deltaT together with KCNQ3 subunits generated functional voltage-gated K+ currents having pharmacological and biophysical properties of heteromeric channels. Collectively, the present results suggest that mutation-induced reduced stability of KCNQ2 subunits may cause epilepsy in neonates.

Highlights

Among neuronal Kϩ currents, the muscarinic-regulated Kϩ current (IKM)3 activates slowly during long-lasting depolarizing inputs and repolarizes the neuronal membrane back toward resting membrane potential [2], limiting repetitive firing and causing spike-frequency adaptation [3]
Functional Characterization of Fusion Proteins between enhanced green fluorescent protein (EGFP) and KCNQ2 Subunits—Fig. 1A shows representative traces of macroscopic Kϩ current recordings from Chinese hamster ovary (CHO) cells transfected with the cDNA encoding for fusion proteins in which the EGFP was placed at the N terminus (EGFP-Q2), or at the C terminus (Q2-EGFP) of the Q2 subunit, as compared with those of CHO cells transfected with the plasmid encoding for EGFP (EGFP) or co-transfected with EGFP and Q2 plasmids (EGFP ϩ Q2)
Both fusion constructs gave rise to Kϩ currents significantly larger than EGFP-transfected control cells, the currents recorded from Q2-EGFP-trasfected cells were smaller than those recorded from EGFP-Q2-transfected cells (Fig. 1B)

Summary

Introduction

Among neuronal Kϩ currents, the muscarinic-regulated Kϩ current (IKM)3 activates slowly during long-lasting depolarizing inputs and repolarizes the neuronal membrane back toward resting membrane potential [2], limiting repetitive firing and causing spike-frequency adaptation [3]. Electrophysiological analysis of these two fusion constructs after 24 – 48 h post-transfection revealed that the EGFP-Q2 2043⌬T mutation failed to give rise to functional voltage-gated Kϩ channels; on the other hand, EGFP-Q2 2513⌬G mutant subunits were able to produce significant amounts of Kϩ currents, these were smaller than those recorded from EGFP-Q2-transfected cells (Fig. 2, A and B).

Results

Conclusion