Fluorescence Microscopy Tools to Study the Heteromeric Assembly of an Ion Channel

Abstract

Hyperpolarization-activated, Cyclic Nucleotide-gated channels, HCN, are membrane protein belonging to the superfamily of voltage gated potassium (Kv). They assemble from identical or similar monomers in tetramer to form “pacemaker” channel in cardiac and nerve cells. Recent studies attributed to the isoform HCN1 a key role in neonatal epileptic encephalopathy as the analyzed patients exhibited single point mutations in the gene encoding for the ion channel. In order to study the impact of these mutations on the HCN1-driven current, we reproduced in vitro the heterozygous condition of the patients by cotransfecting HEK 293 cells with the wt and mutant genes. Co-expression of the wild-type and mutated genes in some cases resulted in an intermediate channel behavior, suggesting that heterotetramerization does occur in our system. In principle, to prove ion channel heteromerization, one can choose among several fluorescence microscopy techniques Nevertheless not all the conventional approaches can be adopted. In this work, we tested different techniques to study the trafficking and the assembly of HCN1: colocalization, Förster Resonance Energy Transfer (FRET), cross-correlation Raster Image Correlation Spectroscopy (ccRICS) and Bimolecular Fluorescence Complementation (BiFC). We will present advantages and limitations of these approaches in the study of wild-type and mutated HCN1 heteromerization.