Ca transport mediated by a synthetic neutral Ca-ionophore in biological membranes

The effect of a synthetic neutral ligand on the Ca²⁺ permeability of several biological membranes has been investigated. The ligand had been previously shown to possess Ca²⁺-ionophoric activities in artificial phospholipid membranes. The neutral ionophore is able to transport Ca²⁺ across the membranes of erythrocytes and sarcoplasmic reticulum, when lipophilic anions such as tetraphenylborate or carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) are present, presumably to facilitate the diffusion of the charged Ca²⁺-ionophore complex across the hydrophobic core of the membrane.In mitochondria, the neutral ionophore promotes the active transport of Ca²⁺ in response to the negative membrane potential generated by respiration, in the presence of the specific inhibitor of the natural carrier ruthenium red.     

Ca2+ transport mediated by a synthetic neutral Ca2+-ionophore in biological membranes

The effect of a synthetic neutral ligand on the Ca²⁺ permeability of several biological membranes has been investigated. The ligand had been previously shown to possess Ca²⁺-ionophoric activities in artificial phospholipid membranes. The neutral ionophore is able to transport Ca²⁺ across the membranes of erythrocytes and sarcoplasmic reticulum, when lipophilic anions such as tetraphenylborate or carbonylcyanide $\text{p-}\text{trifluoromethoxyphenylhydrazone}$ (FCCP) are present, presumably to facilitate the diffusion of the charged Ca²⁺-ionophore complex across the hydrophobic core of the membrane.In mitochondria, the neutral ionophore promotes the active transport of Ca²⁺ in response to the negative membrane potential generated by respiration, in the presence of the specific inhibitor of the natural carrier ruthenium red.     

The presence of membranes or micelles induces structural changes of the myristoylated guanylate-cyclase activating protein-2

Guanylate cyclase-activating proteins (GCAPs) are neuronal Ca²⁺ sensors that play a central role in shaping the photoreceptor light response and in light adaptation through the Ca²⁺-dependent regulation of the transmembrane retinal guanylate cyclase. GCAPs are N-terminally myristoylated, and the role of the myristoyl moiety is not yet fully understood. While protein lipid chains typically represent membrane anchors, the crystal structure of GCAP-1 showed that the myristoyl chain of the protein is completely buried within a hydrophobic pocket of the protein, which stabilizes the protein structure. Therefore, we address the question of the localization of the myristoyl group of GCAP-2 in the absence and in the presence of lipid membranes as well as DPC detergents (as a membrane substitute amenable to solution state NMR). We investigate membrane binding of both myristoylated and nonmyristoylated GCAP-2 and study the structure and dynamics of the myristoyl moiety of GCAP-2 in the presence of POPC membranes. Further, we address structural alterations within the myristoylated N-terminus of GCAP-2 in the presence of membrane mimetics. Our results suggest that upon membrane binding the myristoyl group is released from the protein interior and inserts into the lipid bilayer.     

The effect of cyclic nucleotides and protein phosphorylation on the permeability of human erythrocyte ghosts to certain cations

Summary Preparations of human erythrocyte membranes have been made which are in the form of sealed vesicles and which behave as osmometers on suspension in solutions of simple inorganic salts. Using these preparations the permeability of the membranes to Na⁺, K⁺, Mg²⁺ and Ca²⁺ was measured. Cyclic AMP (but not cyclic GMP) increased the permeability of the membranes to Ca²⁺ with a half maximal effect at a concentration of 25µm but did not affect the permeability to the other ions tested. Phosphorylation of proteins in the erthrocyte membrane lowered the permeability to Ca²⁺ without affecting the permeability to the other ions tested and there was a good correlation between the time course of protein phosphorylation and decrease in Ca²⁺ permeability.It is postulated that the system through which cyclic AMP causes an initial rapid rise in Ca²⁺ permeability followed by increased phosphorylation of membrane proteins and reduced Ca²⁺ permeability may have a widespread occurrence in biological systems and serve to control the concentration of Ca²⁺ in the cytoplasm.     

The influence of Ca2+ on the lateral lipid distribution and phase transition in phosphatidylethanolamine/phosphatidylserine vesicles

The lateral lipid distribution within dipalmitoylphosphatidylethanolamine (DPPE)/dipalmitoylphosphatidylserine (DPPS) vesicle membranes was investigated under the influence of Ca²⁺ using a lipid cross-linking method. To characterize the phase transition in DPPE/DPPS vesicles and to correlate the different phase states of the membrane lipids with the obtained lipid distribution ESR measurements using a fatty acid spin label were carried out. It is shown that Ca²⁺ has a significant influence on the lateral lipid distribution within the fluid phase of the membrane lipids; instead of a slight alternating lipid arrangement in absence of Ca²⁺ due to the electrostatic interaction between the DPPS headgroups after addition of Ca²⁺ a lateral cluster structure is characteristic of the fluid phase.     

Calcium-activated membrane interaction of the islet amyloid polypeptide: implications in the pathogenesis of type II diabetes mellitus

The role played by Ca²⁺ ions in the interaction of the human islet amyloid polypeptide (hIAPP) with model membranes has been investigated by differential scanning calorimetry (DSC) and circular dichroism (CD) experiments. In particular, the interaction of hIAPP and its rat isoform (rIAPP) with zwitterionic dipalmitoyl-phosphatidylcholine (DPPC), negatively charged dipalmitoyl-phosphatidylserine (DPPS) vesicles and with a 3:1 mixtures of them, has been studied in the presence of Ca²⁺ ions. The experiments have evidenced that amorphous, soluble hIAPP assemblies interact with the hydrophobic core of DPPC bilayers. Conversely, the presence of Ca²⁺ ions is necessary to activate a preferential interaction of hIAPP with the hydrophobic core of DPPS membranes. These findings support the hypothesis that an impaired cellular homeostasis of Ca²⁺ ions may promote the insertion of hIAPP into the hydrophobic core of carrier vesicles which is thought to contribute to an eventual intracellular accumulation of β-sheet rich hIAPP aggregates.     

25-Hydroxysterols increase the permeability of liposomes to Ca2+ and other cations

25-Hydroxycholesterol and 25-hydroxy vitamin D-3 increased the permeability of liposomes to Ca²⁺ measured by the arsenazo III encapsulation technique. This effect was sensitive to the lipid composition of the membrane, with changes that decreased the motional freedom of phospholipid acyl chains decreasing Ca²⁺ permeability. The greatest permeability was observed with the zwitter-ionic phospholipids, phosphatidylcholine and phosphatidylethanolamine, whereas the acidic phospholipids, phosphatidylinositol and phosphatidylserine, depressed Ca²⁺ permeability. The effect was not specific for Ca²⁺. Other divalent cations were translocated in the order Mn²⁺ > Mg²⁺  Ca²⁺ ? Sr²⁺  Ba²⁺. The permeability of liposomes to the monovalent cation, Na⁺, was also substantially increased. The effect did not appear to be due to ionophoretic properties of the sterols, and it is suggested that perturbation of the membranes by the polar 25-hydroxyl group may play a role in increasing membrane permeability.     

Palmitic Acid Induces the Opening of a Ca2+-Dependent Pore in the Plasma Membrane of Red Blood Cells: The Possible Role of the Pore in Erythrocyte Lysis

Earlier we found that in the presence of Ca²⁺ palmitic acid (Pal) increases the nonspecific permeability of artificial (planar and liposomal) membranes and causes permeabilization of the inner mitochondrial membrane. An assumption was made that the mechanism of Pal/Ca²⁺-induced membrane permeabilization relates to the Ca²⁺-induced phase separation of Pal and can be considered as formation of fast-tightening lipid pores due to chemotropic phase transition in the lipid bilayer. In this article, we continue studying this pore. We have found that Pal plus Ca²⁺ permeabilize the plasma membrane of red blood cells in a dose-dependent manner. The same picture has been revealed for stearic acid (20 μM) but not for myristic and linoleic acids. The Pal-induced permeabilization of erythrocytic membranes can also occur in the presence of Ba²⁺ and Mn²⁺ (200 μM), but other bivalent cations (200 μM Mg²⁺, Sr²⁺, Ni²⁺, Co²⁺) are relatively ineffective. The formation of Pal/Ca²⁺-induced pores in the erythrocytic membranes has been found to result in the destruction of cells.     

Minocycline chelates Ca<Superscript>2+</Superscript>, binds to membranes,and depolarizes mitochondria by formation of Ca<Superscript>2+</Superscript>-dependent ion channels

Minocycline (an anti-inflammatory drug approved by the FDA) has been reported to be effective in mouse models of amyotrophic lateral sclerosis and Huntington disease. It has been suggested that the beneficial effects of minocycline are related to its ability to influence mitochondrial functioning. We tested the hypothesis that minocycline directly inhibits the Ca²⁺-induced permeability transition in rat liver mitochondria. Our data show that minocycline does not directly inhibit the mitochondrial permeability transition. However, minocycline has multiple effects on mitochondrial functioning. First, this drug chelates Ca²⁺ ions. Secondly, minocycline, in a Ca²⁺-dependent manner, binds to mitochondrial membranes. Thirdly, minocycline decreases the proton-motive force by forming ion channels in the inner mitochondrial membrane. Channel formation was confirmed with two bilayer lipid membrane models. We show that minocycline, in the presence of Ca²⁺, induces selective permeability for small ions. We suggest that the beneficial action of minocycline is related to the Ca²⁺-dependent partial uncoupling of mitochondria, which indirectly prevents induction of the mitochondrial permeability transition.     

Al and Ca Alteration of Membrane Permeability of Quercus rubra Root Cortex Cells

This study was undertaken to quantify the effect of aluminum and calcium on membrane permeability. The influence of Ca²⁺ (0.2-3.7 millimolar) and Al³⁺ (0-3.7 millimolar) on the permeability of root cortical cells of Quercus rubra was measured using three nonelectrolytes (urea, methyl urea, and ethyl urea) as permeators of progressively increasing lipid solubility. Water permeability was also measured. Al³⁺ (a) increased membrane permeability to the nonelectrolytes, (b) decreased the membrane's partiality for lipid permeators, and (c) decreased membrane permeability to water. Ca²⁺ had effects on permeability that were opposite to those of Al³⁺. When Al³⁺ and Ca²⁺ were tested in combination, these opposite effects counteracted each other. The results suggest that Al³⁺ altered the architecture of membrane lipids.     

Insight into the location and dynamics of the annexin A2 N-terminal domain during Ca-induced membrane bridging

Annexin A2 (AnxA2) is a Ca²⁺- and phospholipid-binding protein involved in many cellular regulatory processes. Like other annexins, it is constituted by two domains: a conserved core, containing the Ca²⁺ binding sites, and a variable N-terminal segment, containing sites for interactions with other protein partners like S100A10 (p11). A wealth of data exists on the structure and dynamics of the core, but little is known about the N-terminal domain especially in the Ca²⁺-induced membrane-bridging process. To investigate this protein region in the monomeric AnxA2 and in the heterotetramer (AnxA2-p11)₂, the reactive Cys8 residue was specifically labelled with the fluorescent probe acrylodan and the interactions with membranes were studied by steady-state and time-resolved fluorescence. In membrane junctions formed by the (AnxA2-p11)₂ heterotetramer, the flexibility of the N-terminal domain increased as compared to the protein in solution. In “homotypic” membrane junctions formed by monomeric AnxA2, acrylodan moved to a more hydrophobic environment than in the protein in solution and the flexibility of the N-terminal domain also increased. In these junctions, this domain is probably not in close contact with the membrane surface, as suggested by the weak quenching of acrylodan observed with doxyl-PCs, but pairs of N-termini likely interact, as revealed by the excimer-forming probe pyrene-maleimide bound to Cys8. We present a model of monomeric AnxA2 N-terminal domain organization in “homotypic” bridged membranes in the presence of Ca²⁺.     

Lipid-anchored Synaptobrevin Provides Little or No Support for Exocytosis or Liposome Fusion

SNARE proteins catalyze many forms of biological membrane fusion, including Ca²⁺-triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca²⁺-triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca²⁺-synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-α, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-α may reflect a slower alternative mode of SNARE-mediated membrane fusion.     

Isolation of calcium-transporting lipid from the mitochondrial glycolipoprotein

Summary From the mitochondrial Ca²⁺-transporting glycolipoprotein (GLP) the lipid was isolated which induced Ca²⁺-translocation through bilayer lipid membranes. Electroconductivity of modified phospholipid membranes in the presence of CaCl₂ is increased 150-200 times. At 10-fold CaCl₂ gradient a generation of membrane potential is observed close to its theoretical value. It is shown that the lipid forms separate conductivity channels of 10 and 20 pS in the bilayer. The mode of action of GLP in the membrane is proposed It is assumed that the carbohydrate part of GLP is a selective receptor-accumulator for Ca²⁻, whereas the function of the lipid component consists in forming channels in the bilayer.     

α-Adrenergically mediated changes in membrane lipid fluidity and Ca2+ binding in isolated rat liver plasma membranes

Noradrenaline (0.1–5 μM, in the presence of 5 μM propranolol to block β-receptors), ATP (100 μM) and angiotensin II (0.1 μM), which are thought to increase cytosolic Ca²⁺ concentration by mobilizing Ca²⁺ from internal stores, increased the lipid fluidity as measured by diphenylhexatriene fluorescence polarization in plasma membranes isolated from rat liver. The effect of noradrenaline was dose-dependent and blocked by the α-antagonists phenoxybenzamine (50 μM) and phentolamine (1 μM). The response to a maximal dose of noradrenaline (5 μM) and that to ATP (100 μM) were not cumulative, suggesting that both agents use a common mechanism to alter the membrane lipid fluidity. In contrast, the addition of noradrenaline (5 μM) along with the foreign amphiphile Na⁺-oleate (1–30 μM) resulted in an increase in membrane lipid fluidity which was equivalent to the sum of individual responses to the two agents. In the absence of Mg²⁺, reducing free Ca²⁺ concentration by adding EGTA increased membrane lipid fluidity and abolished the effect of noradrenaline, suggesting that Ca²⁺ is involved in the mechanism by which the hormone exerts its effect on plasma membranes. Noradrenaline (5 μM) and angiotensin II (0.1 μM) also promoted a small release of ⁴⁵Ca²⁺ (16 pmol/mg membrane proteins) from prelabelled plasma membranes. The effect of noradrenaline was suppressed by the α-antagonist phentolamine (5 μM). It is proposed that noradrenaline, via α-adrenergic receptors and other Ca²⁺-mobilizing hormones, increases membrane lipid fluidity by displacing a small pool of Ca²⁺ bound to phospholipids, removing thus the mechanical constraints brought about by this ion.     

Involvement of carboxyl groups in the divalent cation permeability of rat parotid gland basolateral plasma membrane

Divalent cation permeability of rat parotid gland basolateral plasma membranes was examined in dispersed parotid acini (by Ca²⁺ or Mn²⁺ entry) and in isolated basolateral plasma membrane vesicles (BLMV, by⁴⁵Ca²⁺ influx). Mn²⁺ entry (fura2 quenching) was about 1.6 fold higher in internal Ca²⁺ pool-depleted acini (Ca²⁺-depl acini) than in unstimulated cells. Mn²⁺ entry into Ca²⁺-depl acini was increased at external pH>7.4 and decreased at pH<7.4. Pretreatment of Ca²⁺-depl acini with the relatively hydrophobic carboxylic group reagent, N,N-dicyclohexylcarbodiimide (DCCD, 50 M for 30 min) resulted in the inhibition of Mn²⁺ entry into Ca²⁺-depl acini to unstimulated levels. Another hydrophobic carboxyl group reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and the relatively hydrophilic carboxyl group reagents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMCD) did not affect Mn²⁺ entry.Similar to the effects in intact acini, Ca²⁺ influx into BLMV was decreased when the external pH was lowered below 7.4. Also DCCD (5 mM, 30 min), but not EEDQ, decreased (40%) Ca²⁺ influx in BLMV. However, unlike in acini, the hydrophilic reagents, EDC, EAC, and CMCD decreased Ca²⁺ permeability in BLMV and the effects were nonadditive with the decrease induced by DCCD. The aggregate effects of carboxyl group reagents on the Ca²⁺ and Mn²⁺ permeability in BLMV and intact acini, respectively, suggest that a critical carboxyl group (most likely accessible from the cytoplasmic side of the plasma membrane) is involved in divalent cation flux in rat parotid acinar cells.     

A permeability transition in liposomes induced by the formation of Ca/palmitic acid complexes

Formation of palmitic acid/Ca²⁺ (PA/Ca²⁺) complexes was suggested to play a key role in the non-classical permeability transition in mitochondria (NCPT), which seems to be involved in the PA-induced apoptosis of cardiomyocytes. Our previous studies of complexation of free fatty acids (FFA) with Ca²⁺ showed that long-chain (C:16-C:22) saturated FFA had an affinity to Ca²⁺, which was much higher than that of other FFA and lipids. The formation of FFA/Ca²⁺ complexes in the black-lipid membrane (BLM) was demonstrated to induce a nonspecific ion permeability of the membrane. In the present work, we have found that binding of Ca²⁺ to PA incorporated into the membrane of sulforhodamine B (SRB)-loaded liposomes results in an instant release of a part of SRB, with the quantity of SRB released depending on the concentration of PA and Ca²⁺. The pH-optimum of this phenomenon, similar to that of PA/Ca²⁺ complexation, is in the alkaline range. The same picture of SRB release has been revealed for stearic, but not for linoleic acid. Along with Ca²⁺, some other bivalent cations (Ba²⁺, Sr²⁺, Mn²⁺, Ni²⁺, Co²⁺) also induce SRB release upon binding to PA-containing liposomes, while Mg²⁺ turns out to be relatively ineffective. As revealed by fluorescence correlation spectroscopy, the apparent size of liposomes does not alter after the addition of PA, Ca²⁺ or their combination. So it has been supposed that the cause of SRB release from liposomes is the formation of lipid pores. The effect of FFA/Ca²⁺-induced permeabilization of liposomal membranes has several analogies with NCPT, suggesting that both these phenomena are of similar nature.     

Cation Permeability and Selectivity of a Root Plasma Membrane Calcium Channel

Calcium channels in the plasma membrane of root cells fulfill both nutritional and signaling roles. The permeability of these channels to different cations determines the magnitude of their cation conductances, their effects on cell membrane potential and their contribution to cation toxicities. The selectivity of the rca channel, a Ca²⁺-permeable channel from the plasma membrane of wheat (Triticum aestivum L.) roots, was studied following its incorporation into planar lipid bilayers. The permeation of K⁺, Na⁺, Ca²⁺ and Mg²⁺ through the pore of the rca channel was modeled. It was assumed that cations permeated in single file through a pore with three energy barriers and two ion-binding sites. Differences in permeation between divalent and monovalent cations were attributed largely to the affinity of the ion binding sites. The model suggested that significant negative surface charge was present in the vestibules to the pore and that the pore could accommodate two cations simultaneously, which repelled each other strongly. The pore structure of the rca channel appeared to differ from that of L-type calcium channels from animal cell membranes since its ion binding sites had a lower affinity for divalent cations. The model adequately accounted for the diverse permeation phenomena observed for the rca channel. It described the apparent submillimolar K _m for the relationship between unitary conductance and Ca²⁺ activity, the differences in selectivity sequences obtained from measurements of conductance and permeability ratios, the changes in relative cation permeabilities with solution ionic composition, and the complex effects of Ca²⁺ on K⁺ and Na⁺ currents through the channel. Having established the adequacy of the model, it was used to predict the unitary currents that would be observed under the ionic conditions employed in patch-clamp experiments and to demonstrate the high selectivity of the rca channel for Ca²⁺ influx under physiological conditions. Received: 23 August 1999/Revised: 12 November 1999     

Oscillating Ca2+-Induced Channel Activity Obtained in BLM with a Mitochondrial Membrane Component

Oscillations in ion fluxes and membrane potential may be observed in cells and in mitochondria as well. We obtained Ca²⁺-induced oscillations in channel activity in black-lipid membranes reconstituted with hydrophobic components extracted from mitochondria. Mitoplasts prepared from purified rat liver mitochondria were extracted with ethanol followed by Folch extraction and further partial purification by silicic acid chromatography. Channel activity was measured in lipid bilayers formed from bovine brain lipids and 10% cardiolipin with addition of the purified tractions. The conductance with 10 mM Ca²⁺ was 100 pS or its multiples. Ca²⁺ gradients of 4 : 1 induced oscillating channel activity for several hours, with initial open states of 40 s and closed states of 56 s; the open times gradually decreasing to 8.6 s. No channel activity was seen without added fractions. The channel activity was associated with a Ca²⁺-binding lipid, nonpolar, low-molecular-weight fraction that in gel electrophoresis was not stained with Coomassie Blue and did not contain carbohydrate-staining material. ¹H-Nuclear magnetic resonance spectra of the substance showed the presence of aliphatic chains and carbonyls, but the detailed structure remains to be elucidated.     

Induction of a non-specific permeability transition in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca²⁺ uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca²⁺ transport system (Bazhenova et al. J Biol Chem 273:4372–4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96–100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352–1356, 2000; Deryabina et al. J Biol Chem 276:47801–47806, 2001) were very resistant to Ca²⁺ overload. However, exposure of yeast mitochondria to 50–100 μM Ca²⁺ in the presence of the Ca²⁺ ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca²⁺/nH⁺-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca²⁺- ETH129-induced activation of the Ca²⁺/H⁺-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca²⁺ overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319–331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37–51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high phosphate concentrations. None of the above-mentioned substances or conditions induced a mPTP-like pore. It is thus evident that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.     

Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the “fusion peptide” (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca²⁺ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca²⁺ binding to the SARS-CoV-2-FP, the role of Ca²⁺ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca²⁺-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca²⁺-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca²⁺ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca²⁺ ions. These findings provide novel mechanistic insights regarding the role of Ca²⁺ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.