Marco Aquila

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.

Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness.

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder β-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.

Pore Forming Properties of Cecropin-Melittin Hybrid Peptide in a Natural Membrane

The pore forming properties of synthetic cecropin-melittin hybrid peptide (Acetyl-KWKLFKKIGAVLKVL-CONH2; CM15) were investigated by using photoreceptor rod outer segments (OS) isolated from frog retinae obtained by using the whole-cell configuration of the patch-clamp technique. CM15 was applied (and removed) to (from) the OS in ~50 ms with a computer-controlled microperfusion system. Once the main OS endogenous conductance was blocked with light, the OS membrane resistance was ≥1 GΩ, allowing high resolution, low-noise recordings. Different to alamethicines, CM15 produced voltage-independent membrane permeabilisation, repetitive peptide application caused a progressive permeabilisation increase, and no single-channel events were detected at low peptide concentrations. Collectively, these results indicate a toroidal mechanism of pore formation by CM15.

Divalent cations modulate membrane binding and pore formation of a potent antibiotic peptide analog of alamethicin

The Ca(2+) modulation of pore formation (and disaggregation) kinetics of a synthetic analog of alamethicin F50/5 ([l-Glu(OMe)(7,18,19)]), a potent antibiotic peptide, was investigated in situ and in vitro. The in situ experiments consisted in whole-cell recording from isolated retinal rod outer segments (OS), because once blocking the only OS endogenous conductance with saturating light, the current flows entirely through the (exogenous) channels formed by the peptide. The kinetics of current change induced by peptide application and removal (in ∼50ms) on the OS extracellular side was measured in the presence of divalent cations at different concentrations. The in vitro experiments consisted on the divalent cations modulation of [l-Glu(OMe)(7,18,19)] binding to a mimetic OS membrane immobilized on a sensor chip surface, employing surface plasmon resonance spectroscopy (SPR). The presence of even low mM Ca(2+) or Mg(2+) sufficed to increase the [l-Glu(OMe)(7,18,19)] apparent affinity for the mimetic OS membrane up to ∼4-fold, which accelerated the activation of the peptide-induced current in OS by ∼10-fold with respect to low Ca(2+). In situ and in vitro experiments indicate that high concentrations of divalent cations increased also membrane rigidity, contrasting their effect on increasing the pore formation rate.

Cyclic nucleotide mapping of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a central role in the regulation of cardiac and neuronal firing rate, and these channels can be dually activated by membrane hyperpolarization and by binding of cyclic nucleotides. cAMP has been shown to directly bind HCN channels and modulate their activity. Despite this, while there are selective inhibitors that block the activation potential of the HCN channels, regulation by cAMP analogs has not been well investigated. A comprehensive screen of 47 cyclic nucleotides with modifications in the nucleobase, ribose moiety, and cyclic phosphate was tested on the three isoforms HCN1, HCN2, and HCN4. 7-CH-cAMP was identified to be a high affinity binder for HCN channels and crosschecked for its ability to act on other cAMP receptor proteins. While 7-CH-cAMP is a general activator for cAMP- and cGMP-dependent protein kinases as well as for the guanine nucleotide exchange factors Epac1 and Epac2, it displays the highest affinity to HCN channels. The molecular basis of the high affinity was investigated by determining the crystal structure of 7-CH-cAMP in complex with the cyclic nucleotide binding domain of HCN4. Electrophysiological studies demonstrate a strong activation potential of 7-CH-cAMP for the HCN4 channel in vivo. So, this makes 7-CH-cAMP a promising activator of the HCN channels in vitro whose functionality can be translated in living cells.

Mutation in S6 domain of HCN4 channel in patient with suspected Brugada syndrome modifies channel function

Diseases such as the sick sinus and the Brugada syndrome are cardiac abnormalities, which can be caused by a number of genetic aberrances. Among them are mutations in HCN4, a gene, which encodes the hyperpolarization-activated, cyclic nucleotide-gated ion channel 4; this pacemaker channel is responsible for the spontaneous activity of the sinoatrial node. The present genetic screening of patients with suspected or diagnosed Brugada or sick sinus syndrome identified in 1 out of 62 samples the novel mutation V492F. It is located in a highly conserved site of hyperpolarization-activated cyclic nucleotide-gated (HCN)4 channel downstream of the filter at the start of the last transmembrane domain S6. Functional expression of mutant channels in HEK293 cells uncovered a profoundly reduced channel function but no appreciable impact on channel synthesis and trafficking compared to the wild type. The inward rectifying HCN4 current could be partially rescued by an expression of heteromeric channels comprising wt and mutant monomers. These heteromeric channels were responsive to cAMP but they required a more negative voltage for activation and they exhibited a lower current density than the wt channel. This suggests a dominant negative effect of the mutation in patients, which carry this heterozygous mutation. Such a modulation of HCN4 activity could be the cause of the diagnosed cardiac abnormality.

Biophysical Characterization of Antimicrobial Peptides Activity: From In Vitro to Ex Vivo Techniques

Antimicrobial peptides (AMPs) are evolutionarily conserved components of the innate immune defense system of many living organisms varying from prokaryotes to eukaryotes, including humans. Due to their broad-spectrum activity and low level of induced resistance, these short aminoacid sequences represent a novel class of potential antimicrobial agents. Besides the development of anti-bacterial drugs, AMPs constitute ideal molecular models for the design of molecules with wide-ranging nanomedical applications, such as anti-tumorigenic agents and pharmacological tools to cure channelopaties. Several techniques are currently used to shed light on the mechanisms of action of AMPs, ranging from the characterization of the interaction between peptides and biomimetic membranes and/or intracellular targets, to the study of AMPs effects on pathogens, living cells and tissues. Comprehensive and multiscale studies are crucial to design new AMPs and to identify molecules that can boost their activity. In this minireview we summarize the most recent achievements in AMP-characterization, with a special emphasis on the integration of biophysical approaches, which can synergistically help to bridge the gap between in vitro and ex vivo investigations.

Enhanced Patch-Clamp Technique to Study Antimicrobial Peptides and Viroporins, Inserted in a Cell Plasma Membrane with Fully Inactivated Endogenous Conductances

Many short peptides (Table 1) selectively permeabilize the bacteria plasma membrane (Fig. 1), leading to their lyses and death: they are therefore a source of antibacterial molecules, and inspiration for novel and more selective drugs. Another class of short (<100 residues) membrane proteins called viroporins, because they are coded by viral genes (Table 1), permeabilizes the membrane of susceptible cells during infection of by most animal viruses (Carrasco, 1995; Fig. 1). The permeabilization leads to host cell lyses and the release of the virus mass, replicated at host cell expense, to propagate the infection. Detailed knowledge of the permeabilization properties of these proteins would allow to design, for instance, selective blockers of these pores, that would contrast the spread of the viral infection.

Pressure-polished borosilicate pipettes are "universal sealer" yielding low access resistance and efficient intracellular perfusion.

Whole-cell recording is the most widely used configuration of the patch recording technique, mainly because it allows to manipulate the intracellular environment while recording membrane current. However, the patch pipette tapered shank and the small tip opening give high access resistances and preclude efficient exchange between pipette solution and cell cytosol. Independently by the recording configuration, another problem of this technique is to gain consistently tight seals.Here we describe a method to enlarge the pipette shank without affecting the tip opening diameter, through the calibrated combination of heat and air pressure, with a custom-made inexpensive setup. These pressure-polished pipettes give small access resistances and allow for the accommodation of pulled quartz or plastic perfusion tubes very close to the pipette tip (to deliver exogenous molecules into the cytosol with a controlled timing). Finally, we describe a method to consistently attain seals with pipettes made from just one glass type, for a wide variety of cell types, isolated from different amphibian, reptilian, fish, and mammalian tissues, and on artificial membranes composed of many different lipid mixtures.