The common features of tetrameric ion channels and the role of electrostatic interactions

Abstract The three tetrameric voltage-gated K+, Na+, and Ca2+ ion channels share a high number of common structural features. Moreover, they share a common charge distribution over the corresponding molecular components. These common features point to an energetically favored pathway of channel opening. This pathway starts from an ion channel conformation consisting of the activation gate closed and the inactivation gate (i.e., the selectivity filter) open. As a consequence of a depolarizing pulse, the stepwise outward movement of the four S4 helices triggers a series of interlinked electrostatic attractive interactions leading to complete opening of the ion channels. The assumption of a stepwise outward movement of the S4 helices is supported not only by the possibility of accounting for the salient kinetic features of these ion channels with a minimum of two, or at most three, free parameters, but also by a similar experimental stepwise movement of the gating charge

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

Interactions of the cationic lipodepsipeptide syringopeptin 25 A (SP25A) with mercury-supported dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS) and dioeleoylphosphatidic acid (DOPA) self-assembled monolayers (SAMs) were investigated by AC voltammetry in 0.1 M KCl at pH 3, 5.4 and 6.8. SP25A targets and penetrates the DOPS SAM much more effectively than the other SAMs not only at pH 6.8, where the DOPS SAM is negatively charged, but also at pH 3, 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

The GM1 Ganglioside Forms GM1-Rich Gel Phase Microdomains within Lipid Rafts

Abstract: Mercury-supported, self-assembled monolayers of the sole (SAMs) dioleoylphosphatidylcholine (DOPC)and of a raft -forming mixture of DOPC, cholesterol (Chol) and palmitoylsphingomyelin of (59:26:15) mol% composition(PSM) , were investigated by electrochemical impedance spectroscopy (EIS), both in the absence and in the presence of the monosialoganglioside GM1. The impedance spectra of these four SAMs were fitted by a series of parallel combinations of a resistance and a capacitance ( RC meshes) and displayed on plots of ω Z ′ against −ω Z ″, where Z ′ and Z ″ are the in -phase and quadrature components of the impedance and is the angular frequency. A comparison among these ωdifferent impedance spectra points to the formation of GM1-rich gel phase microdomains within the lipid rafts of the DOPC/Chol/PSM mixture, thanks to the unique molecular-level smooth support provided by mercury, whichallows EIS to detect the protruding gel phase microdomains by averaging them over a macroscopically large area

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

AbstractThe 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 Ca2+ and Cd2+, 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 Ca2+ 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

Investigation of Na+,K+-ATPase on a solid supported membrane: the role of acylphosphatase on the ion transport mechanism

AbstractCharge translocation by Na+,K+-ATPase was investigated by adsorbing membrane fragments containing Na+,K+-ATPase from pig kidney on a solid supported membrane (SSM). Upon adsorption, the ion pumps were activated by performing ATP concentration jumps at the surface of the SSM, and the capacitive current transients generated by Na+,K+-ATPase were measured under potentiostatic conditions. To study the behavior of the ion pump under multiple turnover conditions, ATP concentration jump experiments were carried out in the presence of Na+ and K+ ions. Current transients induced by ATP concentration jumps were also recorded in the presence of the enzyme α-chymotrypsin. The effect of acylphosphatase (AcP), a cytosolic enzyme that may affect the functioning of Na+,K+-ATPase by hydrolyzing its acylphosphorylated intermediate, was investigated by performing ATP concentration jumps both in the presence and in the absence of AcP. In the presence of Na+ but not of K+, the addition of AcP causes the charge translocated as a consequence of ATP concentration jumps to decrease by about 50% over the pH range from 6 to 7, and to increase by about 20% at pH 8. Conversely, no appreciable effect of pH upon the translocated charge is observed in the absence of AcP. The above behavior suggests that protons are involved in the AcP-catalyzed dephosphorylation of the acylphosphorylated intermediate of Na+,K+-ATPase

Pre-steady state electrogenic events of Ca2+/H+ exchange and transport by the Ca2+-ATPase.

Native or recombinant SERCA (sarco(endo)plasmic reticulum Ca(2+) ATPase) was adsorbed on a solid supported membrane and then activated with Ca(2+) and ATP concentration jumps through rapid solution exchange. The resulting electrogenic events were recorded as electrical currents flowing along the external circuit. Current transients were observed following Ca(2+) jumps in the absence of ATP and following ATP jumps in the presence of Ca(2+). The related charge movements are attributed to Ca(2+) reaching its binding sites in the ground state of the enzyme (E(1)) and to its vectorial release from the enzyme phosphorylated by ATP (E(2)P). The Ca(2+) concentration and pH dependence as well as the time frames of the observed current transients are consistent with equilibrium and pre-steady state biochemical measurements of sequential steps within a single enzymatic cycle. Numerical integration of the current transients recorded at various pH values reveal partial charge compensation by H(+) in exchange for Ca(2+) at acidic (but not at alkaline) pH. Most interestingly, charge movements induced by Ca(2+) and ATP vary over different pH ranges, as the protonation probability of residues involved in Ca(2+)/H(+) exchange is lower in the E(1) than in the E(2)P state. Our single cycle measurements demonstrate that this difference contributes directly to the reduction of Ca(2+) affinity produced by ATP utilization and results in the countertransport of two Ca(2+) and two H(+) within each ATPase cycle at pH 7.0. The effects of site-directed mutations indicate that Glu-771 and Asp-800, within the Ca(2+) binding domain, are involved in the observed Ca(2+)/H(+) exchange