A Conserved Threonine in the S1-S2 Loop of KV7.2 and KV7.3 Channels Regulates Voltage-Dependent

Supplamentary Material

A conserved threonine in the S1-S2 loop of KV7.2 and KV7.3 channels regulates voltage-dependent activation

Yvonne Füll, Guiscard Seebohm, Holger Lerche, Snezana Maljevic#

Pflügers Archiv - European Journal of Physiology

# Corresponding author

Snezana Maljevic, PhD

Dept. of Neurology and Epileptology

Hertie Institute for Clinical Brain Research,

University of Tübingen, Tübingen, Germany

Email:

Surface expression of KV7.2 and 7.3 WT and mutant channels

Immunocytochemistry. For immunofluorescence staining CHO cells were plated on Poly-D-Lysine-coated coverslips 3 - 5 hours before staining. Afterwards they were fixed for 15 – 20 minutes in 4% PFA, washed with PBS and blocked 1 hour in a PBS solution containing 3% normal goat serum (NGS) and 0.2% Triton-X. The primary antibody (polyclonal rabbit anti-KV7.2, Maljevic et al., 2011) was diluted 1:2500 in PBS with 0.2% Triton and 1% NGS and incubated overnight at 4°C. The secondary AlexaFluor488 coupled goat anti-rabbit antibody (Invitrogen, Darmstadt, Germany) was applied for 1 hour at room temperature. DAPI was used to stain cell nuclei and coverslips were mounted using Mowiol (Sigma) for microscopy.

Biotinylation assay and Western blot analysis.

Biotin labeling and cell surface protein isolation were performed using a Pierce Cell Surface Protein Isolation Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. For Western blot analysis, protein concentration was determined (BCA system, Thermo Fisher Scientific) and 20 µg of protein, preincubated at 65°C for 10 minutes, electrophoresed by SDS-PAGE on 8% polyacrylamide gels. The proteins were transferred onto PVDF membranes (PALL Corporation, Port Washington, NY), Western blotting was performed using a rabbit polyclonal anti-KV7.2 antibody (1:1500) and chemiluminescence detection was done according to the manufacturer's protocol (ECL Western Detection Kit; Amersham Pharmacia Biotech Europe, Freiburg, Germany) to detect 92 kDa large channel proteins. Actin was used as a loading control.

Fig. 1. Immunohistochemical analysis revealed equal distribution for WT and mutant KV7.2/KV7.3 channels in transiently transfected CHO cells. Cells expressing KV7.2 and Kv7.3 (A) or KV7.2-T114A and KV7.3-T144A (B) were stained using an anti-KV7.2 and a secondary Alexa 488 goat anti-rabbit antibody (green), revealing the majority of the detected protein residing in the endoplasmatic reticulum. Nuclei were stained with DAPI (blue).

Fig. 2. Western blot analysis of biotinylated membrane proteins obtained from CHO cells transfected with KV7.2 WT or mutant channels and their coexpressions with KV7.3. An anti-KV7.2 antibody revealed similar bands of the expected size (92 kDa) for both WT and mutant KV7.2 channels, except for the coexpression of KV7.2-T114A and KV7.3-T144A showing a slight reduction of the detected protein in the membrane. The total amount of transfected DNA was kept the same.

Reference:

Maljevic S, Naros G, Yalçin Ö, Blazevic D, Loeffler H, Cağlayan H, Steinlein OK, Lerche H (2011) Temperature and pharmacological rescue of a folding-defective, dominant-negative KV7.2 mutation associated with neonatal seizures. Hum Mutat. 32(10):E2283-93.