Deterministic model of Cav3.1 Ca2+ channel and a proposed sequence of its conformations.

A family of current-time curves of T-type Cav3.1 Ca2+ channels available in the literature is simulated by a kinetic model differing from that used for the interpretation of all salient features of Na+ and Shaker K+ channels by the insertion of a multiplying factor expressing the difference between the working potential ϕ and the reversal potential ϕr. This deterministic model is also used to simulate experimental curves taken from the literature for steady-state 'fast inactivation' and for a gradual passage from fast to 'slow inactivation'. A depolarizing pulse induces fast or slow inactivation depending on whether it lasts 100-500 ms or about 1 min, and is believed to cause a collapse of the central pore near the selectivity filter (SF). A number of features of fast and slow inactivation of Cav3.1 Ca2+ channels are qualitatively interpreted on the basis of a sequence of conformational states. Briefly, the conformation responsible for 'fast inactivation' is assumed to have the activation gate open and the inactivation gate (i.e., the SF) inactive. Immediately after a depolarizing pulse, this conformation is inactive and requires a sufficiently long rest time at a far negative holding potential to recover from inactivation. 'Slow inactivation' is ascribed to a different conformation with the activation gate closed and the SF inactive.

Role of the time dependence of Boltzmann open probability in voltage-gated proton channels.

The modeling and simulation of experimental families of current-time (I-t) curves of dimeric voltage-gated proton channels and of proton-conducting voltage sensing domains (VSDs) with a minimum of free parameters requires the movement of protons to be controlled by the rate of increase of the Boltzmann open probability p over time in passing from the holding to the depolarizing potential. Families of I-t curves of protomers and proton-conducting VSDs can be satisfactorily fitted by the use of a single free parameter expressing the rate constant kp for the increase of p over time. Families of I-t curves of dimeric Hv1 channels can be fitted by a model that assumes an initial proton current I1 flowing along the two monomeric units, while they are still operating separately; I1 is gradually replaced by a slower and more potential-dependent current I2 flowing when the two monomers start operating jointly under the control of the coiled-coil domain. Here too, p is assumed to increase over time with a rate constant kp that doubles in passing from I1 to I2, with fit requiring three free parameters. Chord conductance yields erroneously high gating charges when fitted by the Boltzmann function, differently from slope conductance.

Modeling squid axon Na+ channel by a nucleation and growth kinetic mechanism.

A kinetic model accounting for all salient features of the Na+ channel of the squid giant axon is provided. The model furnishes explanations for the Cole-Moore-like effect, the rising phase of the ON gating current and the slow 'intermediate component' of its decaying phase, as well as the gating charge immobilization. Experimental ON ionic currents are semi-quantitatively simulated by the use of only three free parameters, upon assuming that the Na+ channel opening proceeds along with the stepwise aggregation of its four domains, while they are moving their gating charge outward under depolarizing conditions. The inactivation phase of the ON ionic current is interpreted by a progressive electrostatic attraction between the positively charged 'hinged lid' containing the hydrophobic IFM triad and its receptor inside the channel pore, as the stepwise outward movement of the S4 segments of the Na+ channel progressively increases the negative charge attracting the triad to its receptor. The Na+ channel closing is assumed to proceed by repolarization-induced disaggregation of its domains, accompanied by inward movement of their gating charge. The phenomenon of 'gating charge immobilization' can be explained by assuming that gradual structural changes of the receptor over the time course of depolarization strengthen the interaction between the IFM triad and its receptor, causing a slow release of the gating charge during the subsequent repolarization.

On the interaction of the highly charged peptides casocidins with biomimetic membranes.

Casocidin I and II (CI and CII) are structurally related antimicrobial peptides made of 39 and 31 amino acids, respectively, which derive from natural proteolytic processing of αs2-casein and adopt an ordered α-helical structure in biomimetic membranes. Their putative membrane-permeabilizing activity was investigated at Hg-supported self-assembled monolayers (SAMs) and at tethered bilayer lipid membranes (tBLMs); the latter consisted of a monolayer of 2,3,di-O-phytanyl-sn-glycerol-1-tetraethylene-glycol-d,l-α lipoic acid ester thiolipid (DPTL), with a dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylserine (DOPS) monolayer on top of it. Interaction of CI and CII with these biomimetic membranes was studied by four electrochemical techniques at pH 3, 5.4 and 6.8. Peptide incorporation in tBLMs was attempted via scans of electrochemical impedance spectra. Experiments demonstrated that CI and CII penetrate SAMs as well as the distal DOPC monolayer of DPTL/DOPC tBLMs, but not the proximal phytanyl monolayer, with the only exception of CII at pH 5.4. Conversely, CII permeabilized DPTL/DOPS tBLMs to a moderate extent at all investigated pH values by forming holes across the membrane, but not ion channels. Structural distribution of charged residues seemed to prevent CII from having a hydrophobic side of the α-helix capable of stabilizing a regular ion channel in the lipid matrix.

Modeling squid axon K+ channel by a nucleation and growth kinetic mechanism.

A kinetic model accounting for all salient features of the K+ channel of the squid giant axon, including the rising phase of the ON gating charge and the Cole-Moore effect, is provided. Upon accounting for a significant feature distinguishing K+, Na+ and Ca2+ channels from channel-forming peptides modeled in our previous 2016 BBA paper, the nucleation-and-growth kinetic model developed therein is extended to simulate ON ionic and gating currents of the K+ channel of the squid giant axon at different depolarization potentials by the use of only two free parameters. K+ channel opening is considered to proceed by progressive aggregation of single subunits, while they are moving their gating charge outward under depolarizing conditions within their tetrameric structure; K+ channel closing proceeds in the opposite direction, by repolarization-induced disaggregation of subunits, accompanied by inward movement of their gating charge.

When and how the melittin ion channel exhibits ohmic behavior.

Melittin exhibits an ohmic behavior in a lipid bilayer having a DOPC distal leaflet and interposed between a 2.25nm tetraethyleneoxy chain tethered to a mercury drop and an aqueous solution. This behavior is induced in a pH6.8 buffer solution of 0.8µg/mL melittin by a pretreatment consisting of series of electrochemical impedance spectroscopy measurements at bias potentials varied by 50mV steps over a transmembrane potential range from about +250 to -250mV. This metastable ohmic behavior remains unchanged for hours, even after acidifying the solution to pH3. The midpoint potential E1/2 between the positive and negative peaks of the resulting cyclic voltammogram is almost coincident with that of the ohmic channels gramicidin and syringopeptin 25A and shifts by the same amount toward more positive potentials with a pH decrease from 6.8 to 3. This common cyclic voltammetry behavior is explained by a tilt of the DOPC polar heads around the channel mouth of these three peptides and is simulated by a modelistic approach. The ohmic behavior of melittin is explained by the persistence of the peptide orientation initially assumed at trans-negative potentials even after application of trans-positive ones, at sufficiently high peptide-to-lipid molar ratios.

What Ion Flow along Ion Channels Can Tell us about Their Functional Activity.

The functional activity of channel-forming peptides and proteins is most directly verified by monitoring the flow of physiologically relevant inorganic ions, such as Na⁺, K⁺ and Cl-, along the ion channels. Electrical current measurements across bilayer lipid membranes (BLMs) interposed between two aqueous solutions have been widely employed to this end and are still extensively used. However, a major drawback of BLMs is their fragility, high sensitivity toward vibrations and mechanical shocks, and low resistance to electric fields. To overcome this problem, metal-supported tethered BLMs (tBLMs) have been devised, where the BLM is anchored to the metal via a hydrophilic spacer that replaces and mimics the water phase on the metal side. However, only mercury-supported tBLMs can measure and regulate the flow of the above inorganic ions, thanks to mercury liquid state and high hydrogen overpotential. This review summarizes the main results achieved by BLMs incorporating voltage-gated channel-forming peptides, interpreting them on the basis of a kinetic mechanism of nucleation and growth. Hg-supported tBLMs are then described, and their potential for the investigation of voltage-gated and ohmic channels is illustrated by the use of different electrochemical techniques.

Channel-forming activity of syringopeptin 25A in mercury-supported phospholipid monolayers and negatively charged bilayers.

Interactions of the cationic lipodepsipeptide syringopeptin 25A (SP25A) with mercury-supported dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS) and dioeleoylphosphatidic acid (DOPA) self-assembled monolayers (SAMs) were investigated by AC voltammetry in 0.1M KCl at pH3, 5.4 and 6.8. SP25A targets and penetrates the DOPS SAM much more effectively than the other SAMs not only at pH6.8, where the DOPS SAM is negatively charged, but also at pH3, where it is positively charged just as SP25A. Similar investigations at tethered bilayer lipid membranes (tBLMs) consisting of a thiolipid called DPTL anchored to mercury, with a DOPS, DOPA or DOPC distal monolayer on top of it, showed that, at physiological transmembrane potentials, SP25A forms ion channels spanning the tBLM only if DOPS is the distal monolayer. The distinguishing chemical feature of the DOPS SAM is the ionic interaction between the protonated amino group of a DOPS molecule and the carboxylate group of an adjacent phospholipid molecule. Under the reasonable assumption that SP25A preferentially interacts with this ion pair, the selective lipodepsipeptide antimicrobial activity against Gram-positive bacteria may be tentatively explained by its affinity for similar protonated amino-carboxylate pairs, which are expected to be present in the peptide moieties of peptidoglycan strands.

Mechanism of voltage-gated channel formation in lipid membranes.

Although several molecular models for voltage-gated ion channels in lipid membranes have been proposed, a detailed mechanism accounting for the salient features of experimental data is lacking. A general treatment accounting for peptide dipole orientation in the electric field and their nucleation and growth kinetics with ion channel formation is provided. This is the first treatment that explains all the main features of the experimental current-voltage curves of peptides forming voltage-gated channels available in the literature. It predicts a regime of weakly voltage-dependent conductance, followed by one of strong voltage-dependent conductance at higher voltages. It also predicts values of the parameters expressing the exponential dependence of conductance upon voltage and peptide bulk concentration for both regimes, in good agreement with those reported in the literature. Most importantly, the only two adjustable parameters involved in the kinetics of nucleation and growth of ion channels can be varied over broad ranges without affecting the above predictions to a significant extent. Thus, the fitting of experimental current-voltage curves stems naturally from the treatment and depends only slightly upon the choice of the kinetic parameters.

Channel-forming activity of syringopeptin 25A in mercury-supported lipid bilayers with a phosphatidylcholine distal leaflet.

The channel-forming activity of the lipodepsipeptide syringopeptin 25A (SP25A) was investigated at a tethered bilayer lipid membrane (tBLM) with a dioleoylphosphatidylcholine distal leaflet, anchored to a mercury electrode through a hydrophilic tetraethyleneoxy spacer. SP25A was incorporated in the tBLM from different aqueous solutions by recording a series of impedance spectra over a potential range encompassing non-physiological transmembrane potential (Δϕ) values. Once incorporated, SP25A forms stable ion channels over the narrower range of physiological Δϕ values. Ion flow into and out of the spacer, through the lipid bilayer moiety of the tBLM, was monitored by potential step chronocoulometry and cyclic voltammetry at pH3, 5.4 and 6.8. Potassium ion flow into the hydrophilic spacer along the SP25A channels, during the negative potential scan, proceeds in two stages, except at the higher pH and lower SP25A concentration adopted, where it proceeds in a single stage. In light of the behavior of SP25A single channel currents reported in the literature, the first stage is ascribed to large channels resulting from the aggregation of small ones, while the second more negative stage is associated with the small channels resulting from the disaggregation of the large ones.

Interaction Study of Phospholipid Membranes with an N-Glucosylated ß-Turn Peptide Structure Detecting Autoantibodies Biomarkers of Multiple Sclerosis.

The interaction of lipid environments with the type I' ß-turn peptide structure called CSF114 and its N-glucosylated form CSF114(Glc), previously developed as a synthetic antigenic probe recognizing specific autoantibodies in a subpopulation of multiple sclerosis patients' serum, was investigated by fluorescence spectroscopy and electrochemical experiments using large unilamellar vesicles, mercury supported lipid self-assembled monolayers (SAMs) and tethered bilayer lipid membranes (tBLMs). The synthetic antigenic probe N-glucosylated peptide CSF114(Glc) and its unglucosylated form interact with the polar heads of lipid SAMs of dioleoylphosphatidylcholine at nonzero transmembrane potentials, probably establishing a dual electrostatic interaction of the trimethylammonium  and phosphate groups of the phosphatidylcholine polar head with the Glu5 and His8 residues on the opposite ends of the CSF114(Glc) ß-turn encompassing residues 6-9. His8 protonation at pH 7 eliminates this dual interaction. CSF114(Glc) is adsorbed on top of SAMs of mixtures of dioleoylphosphatidylcholine with sphingomyelin, an important component of myelin, whose proteins are hypothesized to undergo an aberrant N-glucosylation triggering the autoimmune response. Incorporation of the type I' ß-turn peptide structure CSF114 into lipid SAMs by potential scans of electrochemical impedance spectroscopy induces defects causing a slight permeabilization toward cadmium ions. The N-glucopeptide CSF114(Glc) does not affect  tBLMs to a detectable extent.

Can gramicidin ion channel affect the dipole potential of neighboring phospholipid headgroups?

The cyclic voltammetry behavior of a mercury-supported tethered bilayer lipid membrane (tBLM) incorporating gramicidin A was investigated in aqueous 0.1 M KCl at pH 6.8, 5.4 and 3. The distal leaflet of the lipid bilayer consisted of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidic acid or a DOPC/cholesterol mixture. In passing from pH 6.8 to pH 3, the midpoint potential between the negative current peak, due to K(+) inflow into the spacer, and the positive current peak, due to K(+) ejection into the aqueous solution, shifts toward more positive potentials, while the separation between these two peaks decreases. This behavior is interpreted quantitatively on the basis of a model relying on tBLM structural features estimated independently in previous works. The only adjustable parameter is the rate constant for cation translocation across a potential energy barrier located in the hydrocarbon tail region. The behavior is ascribed to a dragging of the lipid headgroups adjacent to the gramicidin channel mouth toward the hydrocarbon tail region, with a resulting decrease in the negative charge of the DOPC phosphate group, or of the DOPS carboxyl group, with decreasing pH.

Channel-forming activity of syringomycin E in two mercury-supported biomimetic membranes.

The lipodepsipeptide syringomycin E (SR-E) interacts with two mercury-supported biomimetic membranes, which consist of a self-assembled phospholipid monolayer (SAM) and of a tethered bilayer lipid membrane (tBLM) separated from the mercury surface by a hydrophilic tetraethyleneoxy (TEO) spacer that acts as an ionic reservoir. SR-E interacts more rapidly and effectively with a SAM of dioleoylphosphatidylserine (DOPS) than with one of dioleoylphosphatidylcholine (DOPC). The proximal lipid monolayer of the tBLM has no polar head region, being linked to the TEO spacer via an ether bond, while the distal monolayer consists of either a DOPC or a DOPS leaflet. The ion flow into or out of the spacer through the lipid bilayer moiety of the tBLM was monitored by potential step chronocoulometry and cyclic voltammetry. With the distal monolayer bathed by aqueous 0.1M KCl and 0.8µM SR-E, an ion flow in two stages was monitored with DOPC at pH3 and 5.4 and with DOPS at pH3, while a single stage was observed with DOPS at pH5.4. This behavior was compared with that already described at conventional bilayer lipid membranes. The sigmoidal shape of the chronocoulometric charge transients points to an aggregation of SR-E monomers forming an ion channel via a mechanism of nucleation and growth. The ion flow is mainly determined by potassium ions, and is inhibited by calcium ions. The contribution to the transmembrane potential from the distal leaflet depends more on the nature of the lipid than that of the ion channel.

The effect of the hydrophilic spacer length on the functionality of a mercury-supported tethered bilayer lipid membrane.

A biomimetic membrane consisting of a thiolipid monolayer tethered to a mercury electrode, with a dioleoylphosphatidylcholine (DOPC) monolayer on top of it, was fabricated. The thiolipid, referred to as DPOL, consisted of an octaethyleneoxy (OEO) chain terminated at one end with a lipoic acid residue and covalently linked at the other end to two phytanyl chains. The functionality of this biomimetic membrane, referred to as a tethered bilayer lipid membrane (tBLM), was tested by incorporating gramicidin and alamethicin and verifying their ion channel activity. Advantages and drawbacks with respect to a tBLM using a thiolipid, referred to as DPTL, with a tetraethyleneoxy (TEO) chain were examined by using electrochemical impedance spectroscopy, potential-step chronocoulometry and cyclic voltammetry. The maximum charge surface density of potassium ions stored in the OEO spacer amounts to 70µCcm(-2), as compared to a charge surface density of 45µCcm(-2) in the TEO spacer. The lipid bilayer moiety of the DPOL/DOPC tBLM is somewhat leakier than that of the DPTL/DOPC tBLM at potentials negative of about -0.65V vs. the saturated calomel electrode. The estimated value of the surface dipole potential of the OEO spacer amounts to -0.180V and is, therefore, smaller than that, -0.230V, of the TEO spacer.

Interaction of mixed-ligand monolayer-protected Au144 clusters with biomimetic membranes as a function of the transmembrane potential.

Understanding the interaction of nanoparticles with cell membranes is a high-priority research area for possible biomedical applications. We describe our findings concerning the interaction of Au144 monolayer-protected clusters (MPCs) with biomimetic membranes and their permeabilizing effect as a function of the transmembrane potential. We synthesized Au144(SCH2CH2Ph)60 and modified the capping monolayer with 8-mercaptooctanoic acid (Au144OctA) or thiolated trichogin (Au144TCG), a channel-forming peptide. The interactions of these MPCs with mercury-supported lipid mono- and bilayers were studied with a combination of electrochemical techniques specifically sensitive to changes in the properties of biomimetic membranes and/or charge-transfer phenomena. Permeabilization effects were evaluated through the influence of MPC uptake on the reduction of cadmium(II) ions. The nature and properties of the Au144 capping molecules play a crucial role in controlling how MPCs interact with membranes. The native MPC causes a small effect, whereas both Au144OctA and Au144TCG interact significantly with the lipid monolayer and show electroactivity. Whereas Au144OctA penetrates the membrane, Au144TCG pierces the membrane with its peptide appendage while remaining outside of it. Both clusters promote Cd(2+) reduction but with apparently different mechanisms. Because of the different way that they interact with the membrane, Au144OctA is more effective in Cd(2+) reduction when interacting with the lipid bilayer and Au144TCG performs particularly well when piercing the lipid monolayer.

Dermcidin, an anionic antimicrobial peptide: influence of lipid charge, pH and Zn2+ on its interaction with a biomimetic membrane.

The mechanism of membrane permeabilization by dermcidin (DCD-1L), an antimicrobial peptide present in human sweat, was investigated at a mercury-supported monolayer of dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylserine (DOPS) and at a mercury-supported tethered bilayer lipid membrane (tBLM) consisting of a thiolipid (DPTL) with a DOPC or DOPS monolayer self-assembled on top of it. In an unbuffered solution of pH 5.4, DCD-1L is almost neutral and permeabilizes a DPTL/DOPS tBLM at transmembrane potentials, ϕtrans, which are physiological. In a pH 7 buffer solution DCD-1L bears two negative charges and has no effect on a DPTL/DOPC tBLM, whereas it permeabilizes a DPTL/DOPS tBLM only outside the physiological ϕtrans range; however, the presence of zinc ion induces DCD-1L to permeabilize the DPTL/DOPS tBLM at physiological ϕtrans values. The effect of zinc ions suggests a DCD-1L conformation with its positive N-terminus embedded in the lipid bilayer and the negative C terminus floating on the membrane surface. This conformation can be stabilized by a zinc ion bridge between the His(38) residue of the C terminus and the carboxyl group of DOPS. Chronocoulometric potential jumps from ϕtrans ≅ +160 mV to sufficiently negative values yield charge transients exhibiting a sigmoidal shape preceded by a relatively long "foot". This behavior is indicative of ion-channel formation characterized by disruption of DCD-1L clusters adsorbed on top of the lipid bilayer, incorporation of the resulting monomers and their aggregation into hydrophilic pores by a mechanism of nucleation and growth.

Mercury-supported biomimetic membranes for the investigation of antimicrobial peptides.

Tethered bilayer lipid membranes (tBLMs) consist of a lipid bilayer interposed between an aqueous solution and a hydrophilic "spacer" anchored to a gold or mercury electrode. There is great potential for application of these biomimetic membranes for the elucidation of structure-function relationships of membrane peptides and proteins. A drawback in the use of mercury-supported tBLMs with respect to gold-supported ones is represented by the difficulty in applying surface sensitive, spectroscopic and scanning probe microscopic techniques to gather information on the architecture of these biomimetic membranes. Nonetheless, mercury-supported tBLMs are definitely superior to gold-supported biomimetic membranes for the investigation of the function of membrane peptides and proteins, thanks to a fluidity and lipid lateral mobility comparable with those of bilayer lipid membranes interposed between two aqueous phases (BLMs), but with a much higher robustness and resistance to electric fields. The different features of mercury-supported tBLMs reconstituted with functionally active membrane proteins and peptides of bacteriological or pharmacological interest may be disclosed by a judicious choice of the most appropriate electrochemical techniques. We will describe the way in which electrochemical impedance spectroscopy, potential-step chronocoulometry, cyclic voltammetry and phase-sensitive AC voltammetry are conveniently employed to investigate the structure of mercury-supported tBLMs and the mode of interaction of antimicrobial peptides reconstituted into them.

Can proton pumping by SERCA enhance the regulatory role of phospholamban and sarcolipin?

The effect of the incorporation of phosphorylated phospholamban (pPLN) and sarcolipin (SLN) in mercury-supported self-assembled lipid monolayers and in lipid bilayers tethered to mercury via a hydrophilic spacer was investigated by voltammetric techniques and electrochemical impedance spectroscopy. It was shown that pPLN and SLN do not permeabilize lipid bilayers toward ions at physiological pH. However, they exert a permeabilizing action toward inorganic monovalent cations such as K(+) and Tl(+), but not toward divalent cations such as Ca(2+) and Cd(2+), following a small decrease in pH. This behavior can be associated with their regulatory action on the Ca-ATPase of the sarcoplasmic reticulum (SERCA). SERCA pumps two Ca(2+) ions from the cytosol to the lumen of the sarcoplasmic reticulum (SR) and two protons in the opposite direction, causing a transient decrease of pH in the immediate vicinity of its cytoplasmic domain. This decrease is expected to activate the liberated pPLN molecules and SLN to make the SR membrane leakier toward K(+) and Na(+) and the SLN ion channel to translocate small inorganic anions, such as Cl(-). The effect of pPLN and SLN, which becomes synergic when they are both present in the SR membrane, is expected to favor a rapid equilibration of ions on both sides of the membrane.

Regioselective electrophilic access to naphtho[1,2-b:8,7-b']- and -[1,2-b:5,6-b']dithiophenes.

A two-step one purification access to dichloronaphtho[1,2-b:8,7-b'] and [1,2-b:5,6-b']dithiophenes using bis-alkylnaphthyl alkynes and phthalimidesulfenyl chloride as starting materials has been developed. The functionalization of the carbon-chlorine bonds allowed further modification of NDT core, broadening the potential of the methodology.