Some amphiphatic compounds cause red blood cells to form small bubbles on their

Question

Some amphiphatic compounds cause red blood cells to form small bubbles on their surface. This process is called crenation and these compounds are referred to as crenators. Other amphiphatic compounds cause the formation of invaginations and pits on the red cell surface. These compounds are called cup formers. Give an explanation for the following observations made at pH 7.4 :

1. compounds of the type Ar(CH2)3N+(CH3)3 are crenators of intact red blood cells, but cup formers with unsealed red blood cells ghosts ("ghosts" are red blood cells which have been lysed to release hemoglobin). Ar is an aromatic moiety.

2. compounds of the type Ar(CH2)3N (CH3)2 are cup formers with both intact red blood cells and unsealed red blood cell ghosts

Explanation / Answer

The effects of amphipathic compounds on cell membrane mechanics were originally documented in classic studies conducted in red blood cells (RBCs), which formed the basis for the bilayer couple hypothesis of membrane shape transitions . Compounds that are negatively charged, such as salicylate and TNP, partition preferentially into the outer bilayer leaflet, generating leaflet expansion and outward membrane bending. Positively charged amphipaths, including CPZ, partition into the negatively charged inner leaflet and generate inward membrane bending. The manifestation of this preferential partitioning is demonstrated by images of RBCs that are crenulated when exposed to salicylate or TNP or cupped when exposed to CPZ .Quantitative models based on area-difference elasticity were developed and later used to explain a range of RBC shape transitions , further refining the bilayer couple hypothesis.

The molecular basis for OHC electromotility and NLC is the transmembrane protein prestin .Prestin is a member of the SLC26 superfamily of anion transporters and is expressed highly and selectively in the plasma membrane of OHCs . Models for the role of a motor protein in electromotility predict that the protein undergoes conformational changes within the plane of the membrane or in a manner that coincides with or induces membrane bending . The models purport that the concerted action of many densely packed protein molecules translates to whole-cell axial deformations.

Evidence that the plasma membrane can act as an allosteric modulator of transmembrane protein function is mounting , and membrane perturbations that affect prestin function such as modulation of membrane cholesterol , tension , or the introduction of amphipaths are likely linked to alterations of prestin-membrane interactions. Bilayer couple effects have been well characterized in mechanosensitive ion channels , and membrane curvature stress has been shown to affect the function of many proteins, including rhodopsin and the Ca2+-activated BK channel . Additionally, membrane curvature has been predicted and demonstrated to affect protein oligomerization, a finding made more relevant to the auditory field following the demonstration of prestin self-association . Changes in curvature can result from alterations in the membrane lateral pressure profile, which is highly dependent on membrane composition and influences the work associated with protein conformational changes ,such as those predicted for prestin. Lateral pressure profile effects are more pronounced for proteins with depth-dependent cross sectional areas. Interestingly, a recent electron microscopy study indicates that prestin is a bullet-shaped molecule with a greater cross sectional area in the membrane's inner leaflet .

Given the effects of amphiphiles on RBC shape and the apparent mechanosensitivity of prestin, it is reasonable to postulate that salicylate, CPZ, and TNP exert their influence on prestin function and electromotility through modulation of membrane curvature. However, understanding these effects requires consideration of the unique architecture and mechanics of the OHC lateral wall, which contains three structures: the plasma membrane, a cortical cytoskeleton, and the subsurface cisternae. Specifically, the OHC cortical cytoskeleton is made up of circumferentially wound actin filaments cross-linked by spectrin filaments oriented along the longitudinal axis of the cell . An additional structure of unknown molecular identity, visible in electron micrographs, connects the plasma membrane to the cytoskeleton at fairly regularly spaced intervals both in the axial and circumferential direction. This structure, referred to as the pillar protein, has been postulated to provide necessary anchor points that allow the OHC plasma membrane to bend in a consistent and regular manner . The cortical cytoskeleton and a positive intracellular (turgor) pressure together help maintain the cylindrical shape of the OHC and greatly influence overall cell mechanics . Although previous models have predicted OHC plasma membrane bending between pillar attachment sites, it remains controversial whether the membrane is free to deform in this manner or instead remains tightly coupled to the underlying cytoskeleton, as discussed in recent reviews .

To investigate the hypothesis that amphipathic compounds induce OHC plasma membrane bending, we employed fluorescence polarization microscopy (FPM), a quantitative microscopy technique capable of measuring fluorophore orientation with respect to the plane of the membrane. We recently developed FPM for use in the quasicylindrical OHC and demonstrated its ability to measure the orientation of pyridinium, 4-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl) (di-8-ANEPPS) in the plasma membrane (60). This novel implementation of FPM required a reformulation of the original theory developed by Axelrod for use in sphered RBCs . Di-8-ANEPPS is a voltage-dependent probe designed to partition into the membrane and align with the polar and nonpolar regions of adjacent lipids . In previous studies of di-8-ANEPPS orientation in the OHC, we found that its absorption transition dipole moment maintains an orientation of ~27

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