G Protein-Coupled Receptors, Essay Example

G protein-coupled receptors (GPCRs) are the largest family of cell membrane-bound receptors in humans^1–5.They are involved with sensing molecules outside the cell, and the subsequent transduction of this signal to activate intracellular signaling pathways. Through this,  cellular responses are then produced^1–7. Given their importance in health and disease and their potential for therapeutic intervention through using small molecules as regulators, GPCRs represent the largest family of druggable targets⁵.At least 50% of the marketable drugs today notably interact with GPCRs ^4,5. However, those drugs target only up to 46 of the approximately 400 druggable, nonsensory GPCRs^2,6. To note, ligands or function are unknown for another 150 ‘orphan’ GPCRs^2,4.

This calls for the testing of libraries of hundreds of thousands to millions of compounds for their binding affinities to GPCR, a process that is currently performed using cell-based, high-throughput screening (HTS) assays^8,9.  Although these platforms yield many hits and lead-like compounds, growing need for miniaturization of drug discovery assays to save chemicals and increasing the throughput demand binding assay platforms with stable, robust, cell-free signaling assemblies that comprises the receptor and appropriate molecular transducer components. These assay platforms can be readily adaptable for applications such as microarray/biochip assay formats⁹.

Historically, cell-free radioligand binding assays were used to identify compounds that target GPCRs^5,6. However, these assays have limitations. One is that its binding affinity data do not tell whether the compound is an agonist or an antagonist^5,6. This assay is also limited by the availability of radiolabeled ligands^5,6 which further makes it relatively expensive^5,6. In the process, here are also some concerns over radioactive waste that is produced and is ultimately produces problematic waste management issues. To add to the problem, some isotopes have short half-lives making the process impractically expensive and wasteful ^5-6. Another possible alternative would be fluorescence base assays. However, this process offers a new problem in labeling with the appropriate fluorophore which makes it useful to only a fraction of GPCR ligands^{5, 6}. On the other hand, Surface Plasmon Resonance or SPR based assays rely on solid support making it useful for short term studies only. Due to this, there is a need to develop rapid, label-free and sensitive GPCR base biosensor to use in cell-free GPCR binding assays. This biosensor is expected to make binding assays more adaptable to microarray format in HTS systems.

Biosensor based on chimeric ion channel coupled receptors (ICCRs), where GPCR is fused to an ion channel (IC) (figure 01) ^10–13, can provide answers to all these requirements. These fusion proteins are able to generate a well-defined,digitizable electrical signal readout in response to binding of biomolecules or pharmacological compounds with the receptor domain of ICCR(GPCR)^10–12. A binding event produces a conformational change which in turn transduces into a change in ion channel gating function^10–12. Hence, the binding of a ligand is transformed into a change of millions of ion flux through the IC, resulting in the amplification of a binding event signal to the ion current in detectable levels (>pA in current)^10–12.Therefore, ICCRs allow highly sensitive detection of analytes.

ICCR base sensor is an analytical tool that is still in its infancy. The generality of ability to make functional ICCRs by using different GPCRs and ICs is already proven^10–14, but several challenges remain. ICCRs need to be in a lipid environment to maintain their native conformation. This means that the use of artificial lipid membrane or ALM needs to be considered for the process to be successful. However, the ALM to be used needs to have these key features to function fully for the process: (a) The composition of lipids in ALM should more closely resemble the natural lipid bilayer to retain the activity of protein to a maximum length of time. (b) ALM should be mechanically, electrically and chemically stable enough for an extended period of time to facilitate the long-term structure-activity studies at sensor development stage and to use as the rugged sensors platform. (c) It should be fluidic enough for easy incorporation of ICCRs in a functionally active state, and flexible enough to  accommodate the conformational change of functioning ICCRs (e.g  hydrophobic matching¹⁵) (d) ALM should be exposed to hydrophilic compartments from both sides of the membrane to accommodate extracellular loops of ICCRs. (e) ALM should have a good electrical sealing (specific membrane resistance >10 M? cm²) to measure current of ICCRs from the noise of leakage current of the membrane.

The tethered bilayer lipid membrane (tBLM) platform is uniquely capable among different types of ALMs to meet these requirements. In tBLM, inner leaflet of the lipid bilayer is covalently attached to a solid surface through tethering molecules (Figure 02) ^16,17. This improves the strength and the durability of tBLMs. Some tBLMs are noted to have strong air stability^18,19 and longer shelf-lives reaching the order of months^16,19–29. tBLMs that do not instantaneously fail when subjected to robust mechanical mixing of solutions over their surface or when challenged with transmembrane potentials of >±500 mV^28,30,31 are also available. Tethering molecules have functional groups that allow bonding to the solid support. On the other end, the hydrophilic tether/ spacer stands erect with the lipid tail making it possible for the maintenance of maximum distance between the solid support and the bilayer. The lipid tails of tethering molecules are incorporated into the bilayer above. As a result, these membranes are exposed to aqueous reservoirs on each side of the bilayer^{19,21,32–36} and therefore can  accommodate trans-membrane proteins and ion channels^{16,22,24,27,37–41}.The formation of tBLM is more predictable and controllable than other ALMs therefore formation of tBLM is highly reproducible. Due to molecular engineering inspired by biology, tBLMs are increasingly able to mimic fundamental properties of natural cell membranes, including two-dimensional fluidity in the liquid crystalline phase¹⁶, and electrical sealing ( membrane capacitance Cm = ~0.5 ?F cm^?2, specific membrane resistance: Rm > 10 M? cm²)^{19,20,22,24,33,42}.

The main confounding factor in tBLM is generating a bilayer design optimal for the intended application. While there are other forms of tBLM that may make this possible, a relatively small proportion of tethered molecules in the sparsely tethered bilayer lipid membranes (stBLMs) allows the inner and outer leaflets to become more symmetric. This leads to lateral transport properties within the stBLM that are presumably closer to those of free bilayer membranes(~8 µm²s^-1 in ditsal leaflet ~3 µm²s^-1 in proximal leaflet for 7:3 tether:dilution in one study⁴³). The ease and adaptability of the stBLM system allows it to be custom-tailored to studies of a variety of membrane-associated protein. According to Cornell (et al), stBLMs with tether densities as low as 1% have been shown to be able to provide mobile lipid spaces that are sufficiently large to permit the insertion of membrane-bound protein fractions of up to ~300 kDa (calculated from the molecular volume (v) assuming a 4-nm-thick membrane with an area/lipid of ~1 nm², where the molecular mass (m) is related to the density (r)) ²⁰. These systems can be reliably formed, quite simply and with comparable results, for a range of different lipids compositions²³ and can be engineered into microchips⁴¹.

Then again, the low grafting density of stBLMs makes membranes less stable and less electrically insulative. The decrease in tethering increase the defects density in the membrane which intern increased tBLM residual conductivity. In stBLMs with very low tether densities, vast areas on the membrane surface containing few or no anchor molecules. Such areas may exhibit increased bilayer undulations, fluctuations of lipid density, and/or direct contact to the solid surface creating transient pores contributing to the increase of defects. But in moderately tethered membranes clustering of the hydrophobic parts of the tether creates these vast areas on the surface containing few or no anchor molecules ⁴⁴.  Liu and Faller demonstrated that the driving force for cluster formation was the interaction between the hydrophobic parts of the immobilized molecular anchors inside the phospholipid milieu and the length of the tether (hydrophilic part) which facilitates cluster formation that allows the hydrophobic parts to freely move and get close to each other⁴⁵. This can be overcome by some extent using short tether molecules. However, this results in an undesirable reduction in the volume of the aqueous compartment used to decouple the bilayer from the solid support limiting  the size of peripheral domain that can be accommodated. Therefore, stBLMs with tethering as the only stabilization strategy will limit the possibility of obtaining an ALM with insulation, stability and fluidity that is required for the development of ICCR sensor.

Polymerization of lipid membranes is another significant stabilization mechanism employed with black lipid membranes (BLMs)^46–48 and planer supported lipid membranes (PSLBs)^48–50. Polymerization leads to  the increase of stability often without compromising electrical insulation of the membrane(depending on the polymerizable lipid used)^47–51. However, this affects the mobility of lipids, thus developing rigid membranes. To resolve such problem, the polymerization of lipid membrane with the use of mixture of polymerizable and non-polymerizable lipids is used⁵². Partial polymerization promote phase segregation, that creates a membrane composed of domains of non-polymerizable lipid dispersed in a poly(lipid) network^48,52.The non-polymerized fluidic domains provide sites for ion channel reconstitution^48,52while Poly(lipids) domains stabilize the membrane. Polymerization also do not compromise the electrical sealing of the membtrane⁵². However, polymerized BLM often becomes less stable making it less suitable for sensor application.

In this proposal, I intend to couple two stabilization mechanism(tethering and fractional poymerization) to get electrically insulated and robust stBLM with enough fluidity to accommodate ICCR constructs. This Partially Polymerize Sparsely Tethered Lipid Membrane (pstBLM), would have poly(lipid) patches in both leaflets that would stabilize both proximal and distal leaflets of the membrane in addition to stabilization of proximal leaflet by tethering. Some of the tethering molecules would be located inside the poly(lipid) patches.  Interaction of tether lipid tail with surrounding poly(lipids) can be stronger than the interaction of lipid molecules in non-polymerized lipid regions. If this is true, stabilization that can be achieved by fractional polymerization can be greater than the cumulative stabilization effect the each stabilization mechanism would introduce to the membrane. Polymerization is also expected to reduce the clustering of tethered molecules by  reducing the chance of hydrophobic parts to freely move and get close to each other. This would reduce the defects in the membranes and improve the electrical insulative properies of the membrane.In this pstBLM interplay of two mechanisms gives us a more efficient way to balance conflicting demands for stability and fluidity of membranes that can better accommodate ICCRs.