HCN2 and HCN4 Isoforms Self-assemble and Co-assemble with Equal Preference to Form Functional Pacemaker Channels

Gina M Whitaker,Damiano Angoli,Hamed Nazzari,Ryuichi Shigemoto,Eric A Accili

doi:10.1074/jbc.m610978200

Abstract

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) "pacemaker" channel subunits are integral membrane proteins that assemble as tetramers to form channels in cardiac conduction tissue and nerve cells. Previous studies have suggested that the HCN2 and HCN4 channel isoforms physically interact when overexpressed in mammalian cells, but whether they are able to co-assemble and form functional channels remains unclear. The extent to which co-assembly occurs over self-assembly and whether HCN2-HCN4 heteromeric channels are formed in native tissue are not known. In this study, we show co-assembly of HCN2 and HCN4 in live Chinese hamster ovary cells using bioluminescence resonance energy transfer (BRET(2)), a novel approach for studying tetramerization of ion channel subunits. Together with results from electrophysiological and imaging approaches, the BRET(2) data show that HCN2 and HCN4 subunits self-assemble and co-assemble with equal preference. We also demonstrate colocalization of HCN2 and HCN4 and a positive correlation of their intensities in the embryonic mouse heart using immunohistochemistry, as well as physical interactions between these isoforms in the rat thalamus by coimmunoprecipitation. Together, these data support the formation of HCN2-HCN4 heteromeric channels in native tissue.

Highlights

That they form heteromeric channels in these tissues (1, 7, 8)
We demonstrate that co-assembly of HCN2 and HCN4 in live Chinese hamster ovary (CHO) cells occurs with equal preference compared with self-assembly, using multiple approaches, including bioluminescence resonance energy transfer (BRET2), a novel approach to study the association of ion channel subunits
We found that the BRET2 ratios determined from cells cotransfected with HCN2-green fluorescent protein (GFP) or HCN4-GFP and Kv1.5-Renilla luciferase (Rluc) were not significantly different from those determined from cells transfected with only Kv1.5-Rluc, indicating that our negative control produced nearly background levels of BRET2

Summary

EXPERIMENTAL PROCEDURES

Molecular Biology—The construction of an HCN2 C-terminal deletion (lacking the cyclic nucleotide binding domain and the remainder of the C terminus distal to it, HCN2⌬CNBD) and another mutant lacking the entire N terminus was described previously (12, 30). Immunocytochemistry, Fluorescence Microscopy with Structured Illumination, and Determination of Pearson Correlation Coefficients—Forty-eight hours after transfection, cells on coverslips were washed briefly with PBS and fixed with 2% paraformaldehyde in PBS for 5 min. After one wash with PBS containing 1% NGS, cells were incubated with primary antibodies for 1 h at room temperature. Cells were subsequently washed with PBS three times and incubated with Alexa-555-tagged goat anti-mouse secondary antibodies (Molecular Probes, Inc., Ontario, Canada) at a dilution of 1:1500 in PBS with 1% NGS plus bovine serum albumin for 1 h at room temperature in the dark. Tissue sections were washed three times in PBS, followed by incubation with Alexa 488 or 555-tagged secondary antibodies raised in donkeys (1:1000; Molecular Probes). Values for Pearson correlation coefficients are presented as means Ϯ S.E. in n number of fields of view

RESULTS

Homomeric and Heteromeric

DISCUSSION