Andrea Saponaro

TRIP8b Regulates HCN1 Channel Trafficking and Gating through Two Distinct C-Terminal Interaction Sites

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels in the brain associate with their auxiliary subunit TRIP8b (also known as PEX5R), a cytoplasmic protein expressed as a family of alternatively spliced isoforms. Recent in vitro and in vivo studies have shown that association of TRIP8b with HCN subunits both inhibits channel opening and alters channel membrane trafficking, with some splice variants increasing and others decreasing channel surface expression. Here, we address the structural bases of the regulatory interactions between mouse TRIP8b and HCN1. We find that HCN1 and TRIP8b interact at two distinct sites: an upstream site where the C-linker/cyclic nucleotide-binding domain of HCN1 interacts with an 80 aa domain in the conserved central core of TRIP8b; and a downstream site where the C-terminal SNL (Ser-Asn-Leu) tripeptide of the channel interacts with the tetratricopeptide repeat domain of TRIP8b. These two interaction sites play distinct functional roles in the effects of TRIP8b on HCN1 trafficking and gating. Binding at the upstream site is both necessary and sufficient for TRIP8b to inhibit channel opening. It is also sufficient to mediate the trafficking effects of those TRIP8b isoforms that downregulate channel surface expression, in combination with the trafficking motifs present in the N-terminal region of TRIP8b. In contrast, binding at the downstream interaction site serves to stabilize the C-terminal domain of TRIP8b, allowing for optimal interaction between HCN1 and TRIP8b as well as for proper assembly of the molecular complexes that mediate the effects of TRIP8b on HCN1 channel trafficking.

Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function.

Significance cAMP regulation of ion channels controls higher brain functions, such as sleep, memory, and cognition. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by the direct binding of cAMP to their cytoplasmic tail and inhibited by the neuronal β-subunit tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), which prevents cAMP binding. Understanding the molecular mechanisms of regulation of this family of ion channels is critical because it pertains to the physiological processes and diseases associated with dysfunctions in the HCN current. Here, we explain the dual regulatory system of HCN2 channels in atomic detail. cAMP and TRIP8b do not compete for the same binding site on the HCN2 cytoplasmic tail; rather, they exert their mutual competition by promoting and stabilizing two different conformational states of the protein. cAMP signaling in the brain mediates several higher order neural processes. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels directly bind cAMP through their cytoplasmic cyclic nucleotide binding domain (CNBD), thus playing a unique role in brain function. Neuronal HCN channels are also regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit that antagonizes the effects of cAMP by interacting with the channel CNBD. To unravel the molecular mechanisms underlying the dual regulation of HCN channel activity by cAMP/TRIP8b, we determined the NMR solution structure of the HCN2 channel CNBD in the cAMP-free form and mapped on it the TRIP8b interaction site. We reconstruct here the full conformational changes induced by cAMP binding to the HCN channel CNBD. Our results show that TRIP8b does not compete with cAMP for the same binding region; rather, it exerts its inhibitory action through an allosteric mechanism, preventing the cAMP-induced conformational changes in the HCN channel CNBD.

Binding of the auxiliary subunit TRIP8b to HCN channels shifts the mode of action of cAMP

Hyperpolarization-activated cyclic nucleotide–regulated cation (HCN) channels generate the hyperpolarization-activated cation current Ih present in many neurons. These channels are directly regulated by the binding of cAMP, which both shifts the voltage dependence of HCN channel opening to more positive potentials and increases maximal Ih at extreme negative voltages where voltage gating is complete. Here we report that the HCN channel brain-specific auxiliary subunit TRIP8b produces opposing actions on these two effects of cAMP. In the first action, TRIP8b inhibits the effect of cAMP to shift voltage gating, decreasing both the sensitivity of the channel to cAMP (K1/2) and the efficacy of cAMP (maximal voltage shift); conversely, cAMP binding inhibits these actions of TRIP8b. These mutually antagonistic actions are well described by a cyclic allosteric mechanism in which TRIP8b binding reduces the affinity of the channel for cAMP, with the affinity of the open state for cAMP being reduced to a greater extent than the cAMP affinity of the closed state. In a second apparently independent action, TRIP8b enhances the action of cAMP to increase maximal Ih. This latter effect cannot be explained by the cyclic allosteric model but results from a previously uncharacterized action of TRIP8b to reduce maximal current through the channel in the absence of cAMP. Because the binding of cAMP also antagonizes this second effect of TRIP8b, application of cAMP produces a larger increase in maximal Ih in the presence of TRIP8b than in its absence. These findings may provide a mechanistic explanation for the wide variability in the effects of modulatory transmitters on the voltage gating and maximal amplitude of Ih reported for different neurons in the brain.

Structure-Function Relation of Phospholamban: Modulation of Channel Activity as a Potential Regulator of SERCA Activity

Phospholamban (PLN) is a small integral membrane protein, which binds and inhibits in a yet unknown fashion the Ca2+-ATPase (SERCA) in the sarcoplasmic reticulum. When reconstituted in planar lipid bilayers PLN exhibits ion channel activity with a low unitary conductance. From the effect of non-electrolyte polymers on this unitary conductance we estimate a narrow pore with a diameter of ca. 2.2 Å for this channel. This value is similar to that reported for the central pore in the structure of the PLN pentamer. Hence the PLN pentamer, which is in equilibrium with the monomer, is the most likely channel forming structure. Reconstituted PLN mutants, which either stabilize (K27A and R9C) or destabilize (I47A) the PLN pentamer and also phosphorylated PLN still generate the same unitary conductance of the wt/non-phosphorylated PLN. However the open probability of the phosphorylated PLN and of the R9C mutant is significantly lower than that of the respective wt/non-phosphorylated control. In the context of data on PLN/SERCA interaction and on Ca2+ accumulation in the sarcoplasmic reticulum the present results are consistent with the view that PLN channel activity could participate in the balancing of charge during Ca2+ uptake. A reduced total conductance of the K+ transporting PLN by phosphorylation or by the R9C mutation may stimulate Ca2+ uptake in the same way as an inhibition of K+ channels in the SR membrane. The R9C-PLN mutation, a putative cause of dilated cardiomyopathy, might hence affect SERCA activity also via its inherent low open probability.

Fusicoccin Activates KAT1 Channels by Stabilizing Their Interaction with 14-3-3 Proteins.

The phytotoxin fusicoccin stabilizes the interaction between KAT1 channels and their regulatory protein 14-3-3, leading to an increase in the number and in the activity of the channels. Plants acquire potassium (K+) ions for cell growth and movement via regulated diffusion through K+ channels. Here, we present crystallographic and functional data showing that the K+ inward rectifier KAT1 (K+ Arabidopsis thaliana 1) channel is regulated by 14-3-3 proteins and further modulated by the phytotoxin fusicoccin, in analogy to the H+-ATPase. We identified a 14-3-3 mode III binding site at the very C terminus of KAT1 and cocrystallized it with tobacco (Nicotiana tabacum) 14-3-3 proteins to describe the protein complex at atomic detail. Validation of this interaction by electrophysiology shows that 14-3-3 binding augments KAT1 conductance by increasing the maximal current and by positively shifting the voltage dependency of gating. Fusicoccin potentiates the 14-3-3 effect on KAT1 activity by stabilizing their interaction. Crystal structure of the ternary complex reveals a noncanonical binding site for the toxin that adopts a novel conformation. The structural insights underscore the adaptability of fusicoccin, predicting more potential targets than so far anticipated. The data further advocate a common mechanism of regulation of the proton pump and a potassium channel, two essential elements in K+ uptake in plant cells.

A reduced mechanical model for cAMP-modulated gating in HCN channels

We developed an in silico mechanical model to analyze the process of cAMP-induced conformational modulations in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which conduct cations across the membrane of mammalian heart and brain cells. The structural analysis reveals a quaternary twist in the cytosolic parts of the four subunits in the channel tetramer. This motion augments the intrinsic dynamics of the very same protein structure. The pronounced differences between the cAMP bound and unbound form include a mutual interaction between the C-linker of the cyclic nucleotide binding domain (CNBD) and the linker between the S4 and S5 transmembrane domain of the channel. This allows a mechanistic annotation of the twisting motion in relation to the allosteric modulation of voltage-dependent gating of this channel by cAMP.

A synthetic peptide that prevents cAMP regulation in mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels

Binding of TRIP8b to the cyclic nucleotide binding domain (CNBD) of mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels prevents their regulation by cAMP. Since TRIP8b is expressed exclusively in the brain, we envisage that it can be used for orthogonal control of HCN channels beyond the central nervous system. To this end, we have identified by rational design a 40-aa long peptide (TRIP8bnano) that recapitulates affinity and gating effects of TRIP8b in HCN isoforms (hHCN1, mHCN2, rbHCN4) and in the cardiac current If in rabbit and mouse sinoatrial node cardiomyocytes. Guided by an NMR-derived structural model that identifies the key molecular interactions between TRIP8bnano and the HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8bnano) which successfully prevented β-adrenergic activation of mouse If leaving the stimulation of the L-type calcium current (ICaL) unaffected. TRIP8bnano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers.

Structure-guided design of a cell penetrating peptide preventing cAMP modulation of HCN channels

The auxiliary subunit TRIP8b prevents cAMP activation of HCN channels by antagonizing its binding to their cyclic-nucleotide binding domain (CNBD). By determining an NMR-derived structure of the complex formed by the HCN2 channel CNBD and a minimal TRIP8b fragment, TRIPnano, we show here a bipartite interaction between the peptide and CNBD which prevents cAMP binding in two ways: through direct competition for binding at the distal C-helix of the CNBD; and through an allosteric reduction in cAMP affinity induced by TRIP8b binding to the CNBD N-bundle loop. TRIPnano abolishes cAMP binding in all three isoforms, HCN1, HCN2 and HCN4 and can be used to prevent cAMP stimulation in native f-channels. Application of TRIP8bnano, or its delivery via a cell-penetrating sequence, in sinoatrial node myocytes, selectively inhibits beta-adrenergic stimulation of the native If current and mimics the physiological concentrations of acetylcholine leading to a 30% reduction in the spontaneus rate of action potential firing.

A light-gated potassium channel for sustained neuronal inhibition

Currently available inhibitory optogenetic tools provide short and transient silencing of neurons, but they cannot provide long-lasting inhibition because of the requirement for high light intensities. Here we present an optimized blue-light-sensitive synthetic potassium channel, BLINK2, which showed good expression in neurons in three species. The channel is activated by illumination with low doses of blue light, and in our experiments it remained active over (tens of) minutes in the dark after the illumination was stopped. This activation caused long periods of inhibition of neuronal firing in ex vivo recordings of mouse neurons and impaired motor neuron response in zebrafish in vivo. As a proof-of-concept application, we demonstrated that in a freely moving rat model of neuropathic pain, the activation of a small number of BLINK2 channels caused a long-lasting (>30 min) reduction in pain sensation.BLINK2 is a light-activated potassium channel for optogenetic inhibition of neuronal activity. Alberio et al. apply the tool in systems as diverse as cultured rat neurons, mouse brain slices, behaving zebrafish and a rat model of neuropathic pain.

Mechanical transduction of cytoplasmic-to-transmembrane-domain movements in a hyperpolarization-activated cyclic nucleotide–gated cation channel

Hyperpolarization-activated cyclic nucleotide–gated cation (HCN) channels play a critical role in the control of pacemaking in the heart and repetitive firing in neurons. In HCN channels, the intracellular cyclic nucleotide–binding domain (CNBD) is connected to the transmembrane portion of the channel (TMPC) through a helical domain, the C-linker. Although this domain is critical for mechanical signal transduction, the conformational dynamics in the C-linker that transmit the nucleotide-binding signal to the HCN channel pore are unknown. Here, we use linear response theory to analyze conformational changes in the C-linker of the human HCN1 protein, which couple cAMP binding in the CNBD with gating in the TMPC. By applying a force to the tip of the so-called “elbow” of the C-linker, the coarse-grained calculations recapitulate the same conformational changes triggered by cAMP binding in experimental studies. Furthermore, in our simulations, a displacement of the C-linker parallel to the membrane plane (i.e. horizontally) induced a rotational movement resulting in a distinct tilting of the transmembrane helices. This movement, in turn, increased the distance between the voltage-sensing S4 domain and the surrounding transmembrane domains and led to a widening of the intracellular channel gate. In conclusion, our computational approach, combined with experimental data, thus provides a more detailed understanding of how cAMP binding is mechanically coupled over long distances to promote voltage-dependent opening of HCN channels.