The trafficking and function of many ion channels are regulated by subunits that do not contribute to the pore-forming core of the channel. Three recent papers reveal a role for cytoplasmic tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b, also known as PEX5R and PEX5Rp) as a regulatory auxiliary subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.

The HCN channel pore, consisting of HCN1–HCN4 subunits, opens at negative membrane potentials and generates the Ih current that regulates intrinsic neuronal excitability. Variability in Ih hasbeen reported and abnormalities in this current are linked to epilepsy. The three groups therefore investigated the modulation of HCN channels. Previous studies, using yeast two-hybrid screening and co-immunoprecipitation (see Further Reading), indicated that TRIP8b interacts with HCN proteins. Here, this association was confirmed in mouse and rat brain extracts. Moreover, proteomic analysis by Zolles et al. indicated that TRIP8b is potentially the major endogenous auxiliary HCN channel subunit in the mammalian brain.

Exons 1–4 of TRIP8b undergo alternative splicing and Lewis et al. and Santoro et al. identified nine TRIP8b splice variants in the rat and mouse brain. When co-expressed with HCN1 in HEK293T cells, Xenopus laevis oocytes or hippocampal neurons these variants differentially upregulated or downregulated Ih. These effects were mediated by changes in HCN channel surface expression resulting from alterations in HCN subunit trafficking.

TRIP8b also altered HCN channel gating. Santoro et al. and Zolles et al. showed that TRIP8b slowed channel opening and accelerated channel closing. Furthermore, Lewis et al. and Santoro et al. found that co-expressing TRIP8b with HCN subunits shifted the membrane potential threshold for HCN channel activation to more hyperpolarized potentials, regardless of the isoform tested.

How does TRIP8b affect channel gating? Cyclic AMP shifts the activation threshold of HCN to less hyperpolarized potentials, suggesting that TRIP8b might counteract this facilitation. Indeed, TRIP8b inhibited the effects of exogenous cAMP on HCN2 and HCN4 channels in excised inside-out patches (in which the inner surface of the cell membrane is exposed to experimental treatments; Zolles et al.). Furthermore, Santoro et al. showed that mutations in cAMP binding sites on HCN1 abolished TRIP8b's effects on gating in intact cells.

Different regions of TRIP8b contribute to its effects. Lewis et al. showed that two sites in TRIP8b's conserved regions bind to distinct sites in HCN1. Zolles et al. found that conserved regions in the amino-terminal core domain are crucial for TRIP8b's effects on gating. Moreover, as shown by Santoro et al., motifs in exons 2 and 5 of the extreme N terminus govern the protein's effects on surface expression.

These studies add HCN channels to the list of ion channels that are regulated by auxiliary subunits and show that specific TRIP8b isoforms can differentially regulate Ih and thus control intrinsic neuronal excitability.