# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 4 Different Types of Tethered-Bilayer Lipid Membranes
### Sparsely-tBLMs or stBLMs
stBMs, composed of diluted thio-anchorlipids mentioned above, have served as good membrane models to improve knowledge of several membrane phenomena. For instance, ion transporters and ion channels are incorporated more effectively and possibly, exhibit improved function when they are incorporated into a stBLM. Compared to dense tBLMs, the functionality of pore-forming toxins such as \(\alpha\)-hemolysin able to spontaneously penetrate from aqueous solution in the membrane after formation of the lipid bilayer, was improved upon insertion into stBLMs, whether formed with an anchor lipid having a larger anchoring group [165] or inserted into bilayers diluted with \(\beta\)ME [124], due to, in both cases, a better sub-membrane hydration. In the latter case, EIS has shown that \(\alpha\)-hemolysin channels in sparsely tethered membranes have the same properties as those formed in free-standing bilayer lipid membranes. Structural information from NR allowed a precise localization of the protein within the stBLM and revealed that the interaction between the channel and the disordered membrane was consistent with the predictions based on the channel's X-ray crystal structure [124]. stBLMs have also been employed to develop models mimicking the complex outer membrane of Gram-negative bacteria with adaptable properties and to achieve membrane platforms for screening of potential antimicrobial agents, like _colistin_, an antibiotic belonging to the polymixin family [151]. Associated with EIS, they have been intensively used to investigate the functional mechanism of \(\beta\)-barrel pore-forming toxins, mainly produced by Gram-positive bacteria, belonging to the family of cholesterol-dependent cytoplasins (CDCs), like _pylosin_, the most important actively secreted virulence factor of _Trueperella pyogenes_, an opportunistic pathogen associated with suppurative infections, such as mastitis, septic arthritis, liver abscessation, pneumonia and endometritis [178]. They have been helpful to understand how the specific interaction that exists at the lipid membrane level, between \(\alpha\)-synuclein--a Parkinson disease-related protein--and the glucocerebrosidase--the enzyme deficient in Gaucher disease--results in an inhibition of glucocerebrosidase, and provides a molecular connection between Parkinson and Gaucher diseases [98]. Very recently, [PERSON] et al. have used stBLMs to investigate the pleiotropic effects of statins attributed to mechanisms independent on the cholesterol-lowering effect like anti-inflammatory, antioxidant, immunomodulatory and anti-proliferative properties. They showed that hydrophobic statin (i.e., simvastatin), by interacting with pre-formed membrane containing 40% of cholesterol in the outer leaflet, prevents pore formation by a CDC pore-forming toxin, namely _pneumolysin_, the virulent factor of _Streptococcus pneumoniae_ responsible for pneumonia, meningitis and septicemia; simvastatin by enhancing clustering of cholesterol-enriched lipid microdomains, would prevent _pneumolysin_ pore formation [179]. The pH-dependent action mechanism of two CDC toxins, _metolysin_ and _vaginolysin_, respectively produced by vaginal bacteria, _Lactobacillus iners_ and _Gardureella vaginalis_, and involved in vaginal dysbiosis, has been also elucidated by using preformed cholesterol-rich stBLMs with perspective application in medical diagnostics [153; 180]. Thanks to EIS and NR studies, stBLMs have also shown that amyloid \(\beta\)-oligomers, well-known to reduce the membrane barrier to ion transport, fully insert into the bilayer and affect both membrane leaflets and membrane properties by inducing lateral heterogeneity. These effects induce ion conductances across artificial membranes, depending on their lipid composition [181]. As neural networks strongly depend on electrochemical gradients to function, these findings shed new light on the development and progression of neurodegenerative diseases, offering stBLMs as a valid platform for the study of the effects of \(\beta\) oligomers.