Anna Moroni

Heteromeric HCN1–HCN4 channels: a comparison with native pacemaker channels from the rabbit sinoatrial node

‘Funny‐’ (f‐) channels of cardiac sino‐atrial node (SAN) cells are key players in the process of pacemaker generation and mediate the modulatory action of autonomic transmitters on heart rate. The molecular components of f‐channels are the hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channels. Of the four HCN isoforms known, two (HCN4 and HCN1) are expressed in the rabbit SAN at significant levels. However, the properties of f‐channels of SAN cells do not conform to specific features of the two isoforms expressed locally. For example, activation kinetics and cAMP sensitivity of native pacemaker channels are intermediate between those reported for HCN1 and HCN4. Here we have explored the possibility that both HCN4 and HCN1 isoforms contribute to the native If in SAN cells by co‐assembling into heteromeric channels. To this end, we used heterologous expression in human embryonic kidney (HEK) 293 cells to investigate the kinetics and cAMP response of the current generated by co‐transfected (HCN4 + HCN1) and concatenated (HCN4‐HCN1 (4–1) tandem or HCN1‐HCN4 (1–4) tandem) rabbit constructs and compared them with those of the native f‐current from rabbit SAN. 4–1 tandem, but not co‐transfected, currents had activation kinetics approaching those of If; however, the activation range of 4–1 tandem channels was more negative than that of the f‐channel and their cAMP sensitivity were poorer (although that of 1–4 tandem channels was normal). Co‐transfection of 4–1 tandem channels with minK‐related protein 1(MiRP1) did not alter their properties. HCN1 and HCN4 may contribute to native f‐channels, but a ‘context’‐dependent mechanism is also likely to modulate the channel properties in native tissues.

Tetramerization dynamics of C-terminal domain underlies isoform-specific cAMP gating in hyperpolarization-activated cyclic nucleotide-gated channels.

Background: HCN2 and HCN4 respond to cAMP, whereas HCN1 does not. Results: The C-linker plus CNBD of HCN2 and HCN4 show cAMP-induced tetramerization, whereas that of HCN1 contains prebound cAMP and is tetrameric. Conclusion: HCN1 does not respond to the addition of cAMP because its CNBD contains cAMP already. Significance: Tetramerization of the C terminus controls ligand gating in HCN channels. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dually activated by hyperpolarization and binding of cAMP to their cyclic nucleotide binding domain (CNBD). HCN isoforms respond differently to cAMP; binding of cAMP shifts activation of HCN2 and HCN4 by 17 mV but shifts that of HCN1 by only 2–4 mV. To explain the peculiarity of HCN1, we solved the crystal structures and performed a biochemical-biophysical characterization of the C-terminal domain (C-linker plus CNBD) of the three isoforms. Our main finding is that tetramerization of the C-terminal domain of HCN1 occurs at basal cAMP concentrations, whereas those of HCN2 and HCN4 require cAMP saturating levels. Therefore, HCN1 responds less markedly than HCN2 and HCN4 to cAMP increase because its CNBD is already partly tetrameric. This is confirmed by voltage clamp experiments showing that the right-shifted position of V½ in HCN1 is correlated with its propensity to tetramerize in vitro. These data underscore that ligand-induced CNBD tetramerization removes tonic inhibition from the pore of HCN channels.

Potassium Ion Channels of Chlorella Viruses Cause Rapid Depolarization of Host Cells during Infection

ABSTRACT Previous studies have established that chlorella viruses encode K+ channels with different structural and functional properties. In the current study, we exploit the different sensitivities of these channels to Cs+ to determine if the membrane depolarization observed during virus infection is caused by the activities of these channels. Infection of Chlorella NC64A with four viruses caused rapid membrane depolarization of similar amplitudes, but with different kinetics. Depolarization was fastest after infection with virus SC-1A (half time [t1/2], about 9 min) and slowest with virus NY-2A (t1/2, about 12 min). Cs+ inhibited membrane depolarization only in viruses that encode a Cs+-sensitive K+ channel. Collectively, the results indicate that membrane depolarization is an early event in chlorella virus-host interactions and that it is correlated with viral-channel activity. This suggestion was supported by investigations of thin sections of Chlorella cells, which show that channel blockers inhibit virus DNA release into the host cell. Together, the data indicate that the channel is probably packaged in the virion, presumably in its internal membrane. We hypothesize that fusion of the virus internal membrane with the host plasma membrane results in an increase in K+ conductance and membrane depolarization; this depolarization lowers the energy barrier for DNA release into the host.

The Potassium Channel KAT1 Is Activated by Plant and Animal 14-3-3 Proteins

14-3-3 proteins modulate the plant inward rectifier K+ channel KAT1 heterologously expressed in Xenopus oocytes. Injection of recombinant plant 14-3-3 proteins into oocytes shifted the activation curve of KAT1 by +11 mV and increased the τon. KAT1 was also modulated by 14-3-3 proteins of Xenopus oocytes. Titration of the endogenous 14-3-3 proteins by injection of the peptide Raf 621p resulted in a strong decrease in KAT1 current (∼70% at –150 mV). The mutation K56E performed on plant protein 14-3-3 in a highly conserved recognition site prevented channel activation. Because the maximal conductance of KAT1 was unaffected by 14-3-3, we can exclude that they act by increasing the number of channels, thus ruling out any effect of these proteins on channel trafficking and/or insertion into the oocyte membrane. 14-3-3 proteins also increased KAT1 current in inside-out patches, suggesting a direct interaction with the channel. Direct interaction was confirmed by overlay experiments with radioactive 14-3-3 on oocyte membranes expressing KAT1.

Engineering of a light-gated potassium channel

An optogenetic tool to silence neurons Potassium channels in the cell membrane open and close in response to molecular signals to alter the local membrane potential. Cosentino et al. linked a light-responsive module to the pore of a potassium channel to build a genetically encoded channel called BLINK1 that is closed in the dark and opens in response to low doses of blue light. Zebrafish embryos expressing BLINK1 in their neurons changed their behavior in response to blue light. Science, this issue p. 707 Blue light opens a channel to silence excitable neurons. The present palette of opsin-based optogenetic tools lacks a light-gated potassium (K+) channel desirable for silencing of excitable cells. Here, we describe the construction of a blue-light–induced K+ channel 1 (BLINK1) engineered by fusing the plant LOV2-Jα photosensory module to the small viral K+ channel Kcv. BLINK1 exhibits biophysical features of Kcv, including K+ selectivity and high single-channel conductance but reversibly photoactivates in blue light. Opening of BLINK1 channels hyperpolarizes the cell to the K+ equilibrium potential. Ectopic expression of BLINK1 reversibly inhibits the escape response in light-exposed zebrafish larvae. BLINK1 therefore provides a single-component optogenetic tool that can establish prolonged, physiological hyperpolarization of cells at low light intensities.

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.

Functional characterisation and subcellular localisation of HCN1 channels in rabbit retinal rod photoreceptors

Gating of voltage‐dependent conductances in retinal photoreceptors is the first step of a process leading to the enhancement of the temporal performance of the visual system. The molecular components underlying voltage‐dependent gating in rods are presently poorly defined. In the present work we have investigated the isoform composition and the functional characteristics of hyperpolarisation‐activated cyclic nucleotide‐gated channels (HCN) in rabbit rods. Using immunocytochemistry we show the expression in the inner segment and cell body of the isoform 1 (HCN1). Electrophysiological investigations show that hyperpolarisation‐activated currents (Ih) can be measured only from the cell regions where HCN1 is expressed. Half‐activation voltage (–75.0 ± 0.3 mV) and kinetics (t1/2 of 101 ± 8 ms at –110 mV and 20 °C) of the Ih in rods are similar to those of the macroscopic current carried by homomeric rabbit HCN1 channels expressed in HEK 293 cells. The homomeric nature of HCN1 channels in rods is compatible with the observation that cAMP induces a small shift (2.3 ± 0.8 mV) in the half‐activation voltage of Ih. In addition, the observation that within the physiological range of membrane potentials, cAMP does not significantly affect the gain of the current‐to‐voltage conversion, may reflect the need to protect the first step in the processing of visual signals from changes in cAMP turnover.

The viral potassium channel Kcv: structural and functional features.

The chlorella virus PBCV‐1 was the first virus found to encode a functional potassium channel protein (Kcv). Kcv is small (94 aa) and basically consists of the M1‐P‐M2 (membrane‐pore‐membrane) module typical of the pore regions of all known potassium channels. Kcv forms functional channels in three heterologous systems. This brief review discusses the gating, permeability and modulation properties of Kcv and compares them to the properties of bacterial and mammalian K+ channels.

The short N-terminus is required for functional expression of the virus-encoded miniature K(+) channel Kcv.

Kcv (K+ Chlorella virus) is a miniature virus‐encoded K+ channel. Its predicted membrane–pore–membrane structure lacks a cytoplasmic C‐terminus and it has a short 12 amino acid (aa) cytoplasmic N‐terminus. Kcv forms a functional channel when expressed in human HEK 293 cells. Deletion of the 14 N‐terminal aa results in no apparent differences in the subcellular location and expression level of the Kcv protein. However, the truncated protein does not induce a measurable current in transfected HEK 293 cells or Xenopus oocytes. We conclude that the N‐terminus controls functional properties of the Kcv channel, but does not influence protein expression.

The proapoptotic influenza A virus protein PB1-F2 forms a nonselective ion channel

Background PB1-F2 is a proapoptotic influenza A virus protein of approximately 90 amino acids in length that is located in the nucleus, cytosol and in the mitochondria membrane of infected cells. Previous studies indicated that the molecule destabilizes planar lipid bilayers and has a strong inherent tendency for multimerization. This may be correlate with its capacity to induce mitochondrial membrane depolarization. Methodology/Principal Findings Here, we investigated whether PB1-F2 is able to form ion channels within planar lipid bilayers and microsomes. For that purpose, a set of biologically active synthetic versions of PB1-F2 (sPB1-F2) derived from the IAV isolates A/Puerto Rico/8/34(H1N1) (IAVPR8), from A/Brevig Mission/1/1918(H1N1) (IAVSF2) or the H5N1 consensus sequence (IAVBF2) were used. Electrical and fluorimetric measurements show that all three peptides generate in planar lipid bilayers or in liposomes, respectively, a barely selective conductance that is associated with stochastic channel type fluctuations between a closed state and at least two defined open states. Unitary channel fluctuations were also generated when a truncated protein comprising only the 37 c-terminal amino acids of sPB1-F2 was reconstituted in bilayers. Experiments were complemented by extensive molecular dynamics simulations of the truncated fragment in a lipid bilayer. The results indicate that the c-terminal region exhibits a slightly bent helical fold, which is stable and remains embedded in the bilayer for over 180 ns. Conclusion/Significance The data support the idea that PB1-F2 is able to form protein channel pores with no appreciable selectivity in membranes and that the c-terminus is important for this function. This information could be important for drug development.