Interaction of the Pacemaker Channel HCN1 with Filamin A

Abstract

Pacemaker channels are encoded by the HCN gene family and are responsible for a variety of cellular functions including control of spontaneous activity in cardiac myocytes and control of excitability in different types of neurons. Some of these functions require specific membrane localization. Although several voltagegated channels are known to interact with intracellular proteins exerting auxiliary functions, no cytoplasmic proteins have been found so far to modulate HCN channels. Through the use of a yeast two-hybrid technique, here we showed that filamin A interacts with HCN1, an HCN isoform widely expressed in the brain, but not with HCN2 or HCN4. Filamin A is a cytoplasmic scaffold protein with actin-binding domains whose main function is to link transmembrane proteins to the actin cytoskeleton. Using several HCN1 C-terminal constructs, we identified a filamin A-interacting region of 22 amino acids located downstream from the cyclic nucleotide-binding domain; this region is not conserved in HCN2, HCN3, or HCN4. We also verified by immunoprecipitation from bovine brain that the filamin A-HCN1 interaction is functional in vivo. In filamin A-expressing cells (filamin ؉ ), HCN1 (but not HCN4) channels were expressed in hot spots, whereas they were evenly distributed on the membrane of cells lacking filamin A (filamin ؊ ) indicating that interaction with filamin A affects membrane localization. Also, in filamin ؊ cells the gating kinetics of HCN1 were strongly accelerated relative to filamin ؉ cells. The interaction with filamin A may contribute to localizing HCN1 channels to specific neuronal areas and to modulating channel activity.

HCN Pacemaker Channel Activation Is Controlled by Acidic Lipids Downstream of Diacylglycerol Kinase and Phospholipase A2

“…1, available at www.jneurosci.org as supplemental material)], they do so without causing any major alteration to the channel architecture as reported by unaltered ion permeation (data not shown). These findings both confirm the conclusion that cAMP interaction with the CNBD is not involved in the 4␤PMA facilitation and reveal that the domains required to transduce the effect of 4␤PMA do not include any of the motifs that have been shown to be involved in intermolecular interac- tions with cytoplasmic scaffolding or gating modifier proteins (Gravante et al, 2004;Kimura et al, 2004;Santoro et al, 2004;Zong et al, 2005).…”

Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR

“…However, FLNs are now known to directly interact with membrane-spanning proteins including ion channels, adhesion molecules, and G protein-coupled receptors (36)(37)(38)(39)(40)(41)(42)54). Based on these studies, it is clear that FLNs stabilize their binding partners at the cell surface.…”

Formation of heteromeric hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the hippocampus is regulated by developmental seizures

“…Whereas the post-translational regulation of the HCN channel molecules is not fully understood, recent evidence supports activity-dependent subcellular distribution of the HCN1 subunit (Bender et al, 2004) and interaction of the channels with scaffolding proteins (Gravante et al, 2004). Potentially, seizure activity may alter glycosylation or other post-translational editing of the HCN isoform molecules that will influence their cellular fate.…”