Francesco Tadini-Buoninsegni

Understanding membrane transport processes such as ion occlusion reactions of ion pumps and transporters and the ion gating of channels requires knowledge of lipid bilayer electrostatics. A simple example of the effect of membrane electrostatics on ion transport is the much higher permeability of the membrane to hydrophobic anions, such as tetraphenylborate (TPB^-), compared to hydrophobic cations, such as tetraphenylphosphonium (TPP⁺) or tetraphenylarsonium (TPA⁺). This has been attributed to the membrane dipole potential, of which a major contributor has been determined to be oriented water dipoles in the lipid headgroup region of the membrane. From the ratio of the TPB^- to TPP⁺ or TPA⁺ conductances, the magnitude and polarity of the dipole potential can be estimated. Using the voltage-sensitive dye RH421 in conjunction with the stopped-flow technique and solid-supported membrane electrophysiology here we show that the transport of these ions is not simply a diffusion through the membrane but rather occurs in jumps between discrete binding sites within the membrane. The hydrophobic anion TPB^- causes much greater RH421 spectral changes than TPA⁺. This could be explained by a combination of a stronger interaction of TPB^- with RH421 and a deeper binding of TPB^- within the membrane compared to TPA⁺. The experimental methods, used here for the first time to study the kinetics of ion transport across membranes, are potentially applicable to investigations of the membrane permeability of charged drug molecules, in particular anticancer agents.

Sarcoplasmic reticulum Ca ²⁺ ‐ATPase (SERCA) is a key protein involved in calcium homeostasis and the pivotal role of Ca ²⁺ in a plethora of biological activities makes SERCA a powerful biological target. The intertwining between Ca ²⁺ signalling and reactive oxygen species (ROS) has drawn much attention to investigate the photodynamic effect that photosensitizers (PSs), prodrugs that are light‐activated to produce ROS in photodynamic therapy (PDT), pursue on this important target. However, only limited classes of PSs were considered so far, with most of them being porphyrin‐based scaffolds, without considering, to the best of our knowledge, the important family of ruthenium(II) polypyridyl complexes (RPCs), which still attracts increasing interest in PDT thanks to its unique and versatile chemical‐physical repertoire. With the goal of exploring the potential arising from considering SERCA a possible target for highly effective RPC‐based PSs, herein we studied the photodynamic effect on this protein of [Ru(dppn) ₂ (bpy‐morph)](PF ₆ ) ₂ (RuPS) (dppn = benzo[ i ]dipyrido[3,2‐ a :2′,3′‐ c ]phenazine, bpy‐morph = 2,2′‐bipyridine‐4,4′‐diylbis(morpholinomethanone), where two π‐expansive dppn units guarantees a high ¹ O ₂ sensitization ( ϕ _Δ = 0.54 ± 0.06 in CH ₃ CN). To this aim, the solid supported membrane (SSM) biosensing method was employed, equipped with a LED light ( λ _em = 434 nm, 30 W) to perform irradiation experiments. Light activation of RuPS (10 μM) induced a prompt decrease (30%) of Ca ²⁺ translocation by SERCA, leading to the almost complete loss of residual current after 15 min irradiation (more than 90% decrease). The inhibition of SERCA was then evaluated in vivo in C2C12 myotubes, chosen as a model in skeletal muscle biology. Upon establishing cellular internalization via confocal microscopy, light‐activated RuPS was found to enhance the cytosolic calcium concentration up to 1.5‐fold‐higher than thapsigargin, used as positive control. Moreover, the differentiated phenotype of myotubes was reverted by light‐activated RuPS, an effect which was likely related to the impaired activity of this key protein.

Aim: obtain a field-deployable portable box for the online detection of heavy metals in water through an automatable software-assisted procedure and square-wave anodic stripping voltammetry.

ATP8A2 is a mammalian P4‐ATPase (flippase) that translocates the negatively charged lipid substrate phosphatidylserine from the exoplasmic leaflet to the cytoplasmic leaflet of cellular membranes. Using an electrophysiological method based on solid supported membranes, we investigated the electrogenicity of specific reaction steps of ATP8A2 and explored a potential phospholipid translocation pathway involving residues with positively charged side chains. Changes to the current signals caused by mutations show that the main electrogenic event occurs in connection with the release of the bound phosphatidylserine to the cytoplasmic leaflet and support the hypothesis that the phospholipid interacts with specific lysine and arginine residues near the cytoplasmic border of the lipid bilayer during the translocation and reorientation required for insertion into the cytoplasmic leaflet.

The sarco(endo)plasmic reticulum Ca²⁺−ATPase (SERCA) hydrolyzes ATP to transport Ca²⁺ from the cytoplasm to the sarcoplasmic reticulum (SR) lumen, thereby inducing muscle relaxation. Dysfunctional SERCA has been related to various diseases. The identification of small‐molecule drugs that can activate SERCA may offer a therapeutic approach to treat pathologies connected with SERCA malfunction. Herein, we propose a method to study the mechanism of interaction between SERCA and novel SERCA activators, i. e. CDN1163, using a solid supported membrane (SSM) biosensing approach. Native SR vesicles or reconstituted proteoliposomes containing SERCA were adsorbed on the SSM and activated by ATP concentration jumps. We observed that CDN1163 reversibly interacts with SERCA and enhances ATP‐dependent Ca²⁺ translocation. The concentration dependence of the CDN1163 effect provided an EC₅₀=6.0±0.3 μM. CDN1163 was shown to act directly on SERCA and to exert its stimulatory effect under physiological Ca²⁺ concentrations. These results suggest that CDN1163 interaction with SERCA can promote a protein conformational state that favors Ca²⁺ release into the SR lumen.

P-type ATPases are a large family of membrane transporters that are found in all forms of life. These enzymes couple ATP hydrolysis to the transport of various ions or phospholipids across cellular membranes, thereby generating and maintaining crucial electrochemical potential gradients. P-type ATPases have been studied by a variety of methods that have provided a wealth of information about the structure, function, and regulation of this class of enzymes. Among the many techniques used to investigate P-type ATPases, the electrical method based on solid supported membranes (SSM) was employed to investigate the transport mechanism of various ion pumps. In particular, the SSM method allows the direct measurement of charge movements generated by the ATPase following adsorption of the membrane-bound enzyme on the SSM surface and chemical activation by a substrate concentration jump. This kind of measurement was useful to identify electrogenic partial reactions and localize ion translocation in the reaction cycle of the membrane transporter. In the present review, we discuss how the SSM method has contributed to investigate some key features of the transport mechanism of P-type ATPases, with a special focus on sarcoplasmic reticulum Ca²⁺-ATPase, mammalian Cu⁺-ATPases (ATP7A and ATP7B), and phospholipid flippase ATP8A2.

Cancer is a multifactorial family of diseases that is still a leading cause of death worldwide. More than 100 different types of cancer affecting over 60 human organs are known. Chemotherapy plays a central role for treating cancer. The development of new anticancer drugs or new uses for existing drugs is an exciting and increasing research area. This is particularly important since drug resistance and side effects can limit the efficacy of the chemotherapy. Thus, there is a need for multiplexed, cost-effective, rapid, and novel screening methods that can help to elucidate the mechanism of the action of anticancer drugs and the identification of novel drug candidates. This review focuses on different label-free bioelectrochemical approaches, in particular, impedance-based methods, the solid supported membranes technique, and the DNA-based electrochemical sensor, that can be used to evaluate the effects of anticancer drugs on nucleic acids, membrane transporters, and living cells. Some relevant examples of anticancer drug interactions are presented which demonstrate the usefulness of such methods for the characterization of the mechanism of action of anticancer drugs that are targeted against various biomolecules.

The transient receptor potential akyrin type-1 (TRPA1) is a non-selective cation channel playing a pivotal role in pain sensation and neurogenic inflammation. TRPA1 channels expressed in the central nervous system (CNS) have a critical role in the modulation of cortical spreading depression (CSD), which is a key pathophysiological basis of migraine pain. ADM_09 is a recently developed lipoic acid-based TRPA1 antagonist that is able to revert oxaliplatin-induced neuropathic pain and inflammatory trigeminal allodynia. In this context, aiming at developing drugs that are able to target TRPA1 channels in the CNS and promote an antioxidant effect, permeability across the blood–brain barrier (BBB) represents a central issue. Niosomes are nanovesicles that can be functionalized with specific ligands selectively recognized by transporters expressed on the BBB. In this work, the activity of ADM_09 on neocortex cultures was studied, and an efficient formulation to cross the BBB was developed with the aim of increasing the concentration of ADM_09 into the brain and selectively delivering it to the CNS rapidly after parenteral administration.

Superparamagnetic iron oxide nanoparticles (SPIONs) are widely used in various biomedical applications, such as diagnostic agents in magnetic resonance imaging (MRI), for drug delivery vehicles and in hyperthermia treatment of tumors. Although the potential benefits of SPIONs are considerable, there is a distinct need to identify any potential cellular damage associated with their use. Since human ether à go-go-related gene (hERG) channel, a protein involved in the repolarization phase of cardiac action potential, is considered one of the main targets in the drug discovery process, we decided to evaluate the effects of SPIONs on hERG channel activity and to determine whether the oxidation state, the dimensions and the coating of nanoparticles (NPs) can influence the interaction with hERG channel. Using patch clamp recordings, we found that SPIONs inhibit hERG current and this effect depends on the coating of NPs. In particular, SPIONs with covalent coating aminopropylphosphonic acid (APPA) have a milder effect on hERG activity. We observed that the time-course of hERG channel modulation by SPIONs is biphasic, with a transient increase (∼20% of the amplitude) occurring within the first 1–3 min of perfusion of NPs, followed by a slower inhibition. Moreover, in the presence of SPIONs, deactivation kinetics accelerated and the activation and inactivation I–V curves were right-shifted, similarly to the effect described for the binding of other divalent metal ions (e.g. Cd²⁺ and Zn²⁺). Finally, our data show that a bigger size and the complete oxidation of SPIONs can significantly decrease hERG channel inhibition. Taken together, these results support the view that Fe²⁺ ions released from magnetite NPs may represent a cardiac risk factor, since they alter hERG gating and these alterations could compromise the cardiac action potential.

Phospholipid flippases (P4-ATPases) utilize ATP to translocate specific phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining transmembrane lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the P-type ATPase protein family, which also encompasses the ion transporting P2-ATPases: Ca²⁺-ATPase, Na⁺,K⁺-ATPase, and H⁺,K⁺-ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of ATP and the negatively charged lipid substrate phosphatidylserine, whereas only a diminutive current was generated with the lipid substrate phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic lipid translocation. It is concluded that the electrogenicity is associated with a step in the ATPase reaction cycle directly involved in translocation of the lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases. Significance Phospholipid flippases constitute the largest subfamily of P-type ATPases and have in eukaryotic organisms evolved as a central transport system for selective translocation of phospholipids across biological membranes to generate membrane lipid asymmetry, a property essential for numerous cellular processes. The importance of flippases is highlighted by severe neurological disorders and liver diseases caused by flippase dysfunction in humans. The electrogenicity of phospholipid transport by flippases has not previously been explored. We demonstrated that phosphatidylserine translocation by the flippase ATP8A2 generates electrical current, resulting from specific steps in the flippase reaction cycle moving the charged lipid head group between the membrane bilayer leaflets, and that no charged substrate is being countertransported. These findings unravel key features of phospholipid flippases.