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 voltage-gated 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.
Highlights
The hyperpolarization activated pacemaker current If/ Ih has an established role in underlying generation and neurotransmitter-mediated modulation of cardiac pacemaker activity [1] and control of excitabilty and other functions, including integration of synaptic activity and modulation of synaptic strength, in several different types of neurons [2,3]
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
By use of yeast two-hybrid technique, here we show that filamin A interacts with HCN1, an HCN isoform widely expressed in the brain, but not with HCN2 or HCN4
Summary
The hyperpolarization activated pacemaker current If/ Ih has an established role in underlying generation and neurotransmitter-mediated modulation of cardiac pacemaker activity [1] and control of excitabilty and other functions, including integration of synaptic activity and modulation of synaptic strength, in several different types of neurons [2,3]. HCN1, the first HCN isoform to be cloned [6], is extensively expressed in the brain, with a selective distribution is specific brain areas It is expressed for example in the layer 5 neocortical neurons, in the CA1 and, to a lower degree, CA3 hippocampal regions, in the molecular cell layer of the cerebellum, and in the superior colliculus [7,8,9,3]. The aim of this work was to investigate if HCN1 channels interact with partner proteins in the brain and to check if existing associations with any such proteins have a role in specific cellular localization of the channels and modulation of channel function. We found that HCN1 associates with several proteins and with filamin A, a high molecular mass cytoskeletal protein known to anchor different transmembrane proteins to the actin cytoskeleton and to act as a scaffold for various signaling proteins
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