An approach to the research on ion and water properties in the interphase between the plasma membrane and bulk extracellular solution
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In vivo, cells are immersed in an extracellular solution that contains a variety of bioactive substances including ions and water. Classical electrophysiological analyses of epithelial cells in the stomach and small intestine have revealed that within a distance of several hundred micrometers above their apical plasma membrane, lies an extracellular layer that shows ion concentration gradients undetectable in the bulk phase. This “unstirred layer”, which contains stagnant solutes, may also exist between the bulk extracellular solution and membranes of other cells in an organism and may show different properties. On the other hand, an earlier study using a bacterial planar membrane indicated that H+ released from a transporter migrates in the horizontal direction along the membrane surface much faster than it diffuses vertically toward the extracellular space. This result implies that between the membrane surface and unstirred layer, there is a “nanointerface” that has unique ionic dynamics. Advanced technologies have revealed that the nanointerface on artificial membranes possibly harbors a highly ordered assembly of water molecules. In general, hydrogen bonds are involved in formation of the ordered water structure and can mediate rapid transfer of H+ between neighboring molecules. This description may match the phenomenon on the bacterial membrane. A recent study has suggested that water molecules in the nanointerface regulate the gating of K+ channels. Here, the region comprising the unstirred layer and nanointerface is defined as the interphase between the plasma membrane and bulk extracellular solution (iMES). This article briefly describes the physicochemical properties of ions and water in the iMES and their physiological significance. We also describe the methodologies that are currently used or will be applicable to the interphase research.
KeywordsUnstirred layer Nanointerface Bulk extracellular fluid Ions Membrane Water
The iMES does not perfectly correspond to a “biointerface” or “biological interphase” that is likely to include the cellular plasma membrane in addition to the unstirred layer and a nanointerface as documented in the literature . In this review, we focus on ions and water in the iMES and briefly summarize their unique properties and possible physiological significance. Moreover, we describe several techniques and methodologies that have been applied or will be applicable to analysis of the iMES. Some of the topics were presented at the symposium “The 93rd Annual Meeting of the Physiological Society of Japan” (March 22–24, 2016).
The unstirred layer, which is mostly observed above the epithelial apical membranes exposed to an extracellular fluid in a lumen, contains not only solutes including ions, secretory proteins, and peptides, but also structural elements such as glycans and extracellular regions of ion channels, transporters, and receptors (Fig. 1) [2, 3]. This region is likely to serve as a diffusion barrier, allowing ions to display physicochemical behaviors that are undetectable in the bulk phase [4, 5, 6]. In this context, the H+ parameter in the digestion system has been characterized well. In the stomach, a layer of 100- to 300-µm thickness above the plasma membrane of the epithelial cells has a pH gradient from 2 to 7 . This interphase protects the epithelial surface from damage by the strong acid (pH ≈ 1) of the bulk fluid. The small intestine has a ~500-µm unstirred layer, which has pH of 6–7 and shields the epithelial cells from enteral contents whose pH is approximately neutral [7, 8, 9]. The cells take up nutrition via membrane-integral transporters that carry H+ and peptides together. The excess H+ in the layer likely acts as a driving force for the transporters and facilitates absorption of nutrition. These pH properties seem to be maintained at least partially by the convoluted surface of the mucosa, the glycans attached to membrane proteins, and the mucus [3, 6].
It is reasonable to hypothesize that the unstirred layer may cover a variety of cells including floating immune cells and scatteredly distributed fibroblasts in an organism. In this layer, not only H+ but also other ions may show profiles different from those in the bulk phase. This idea is partially supported by one study involving an artificially fabricated lipid-bilayer membrane . In that study, two chambers, each of which contained any one or two of K+, Na+, H+, and Ca2+ and the corresponding ionophore(s), were separated by the membrane. The ion concentrations in one chamber were set to greatly exceed those in the other. As a consequence, ion concentration gradients were constantly detected in the space located within several hundred micrometers from the membrane surface, reminiscent of the unstirred layer. Thickness of the space depends on the magnitude of the diffusion coefficient of an ion. In vivo, individual cell types have different sets of ion transport machinery, and therefore may have an unstirred layer of variable properties and thickness. It will be worthwhile to elucidate how different profiles of the unstirred layer are established and how they influence cellular and tissue functions.
Water structure in the nanointerface is also investigated by frequency modulation-AFM (FM-AFM) that employs a sharp cantilever with a tip diameter of several nanometers as a force sensor. In this method, a piconewton force can be detected at subnanometer spatial resolution. This excellent performance has enabled visualization of a distribution of water with its one layer sensitivity at the interface as follows . Fukuma et al. analyzed water properties in the interface on a dipalmitoylphosphatidylcholine (DPPC) bilayer using FM-AFM and revealed two hydration layers with marked force emergence. The precise 3D analyses of these layers then visualized their corrugations, which likely mirror the architecture of the lipid headgroups of DPPC. This patterned structure was stably detectable throughout the membrane. The authors suggest that the hydration layers can serve as an energy barrier for the ions and proteins approaching the membrane as well as a component controlling their physicochemical dynamics in the interface .
Identification of the strong interaction of water molecules with the MPC polymer brush by SFG spectroscopy and detection of the particular hydration layers on a DPPC membrane by FM-AFM imply existence of a highly ordered water structure in the nanointerface (see Fig. 4a). This profile may be involved in interfacial ion dynamics, which is clearly different from the unstirred-layer’s physicochemical properties governed primarily by a diffusion process. Heberle et al. extracted a planar membrane expressing a H+-secreting transporter, bacteriorhodopsin, from Halobacterium salinarum and examined the behavior of the ion released from the transporter. In this assay, Cys36, a residue localized in the transporter’s intracellular loop in close vicinity of the outer leaflet of the membrane, was bound to an H+-sensitive probe, fluorescein, whereas the extracellular solution exposed to the membrane also contained the other probe, pyramine. H+ released in response to illumination migrates to the bulk extracellular compartment as well as along the outer leaflet and then inner leaflet of the bacterial planar membrane. Monitoring of the change of light intensity of the two different probes revealed that the released H+ diffused along the membrane surface much faster than toward the bulk phase . Similar rapid H+ movement is also observed in ice, which consists of water molecules assembled with highly ordered hydrogen bond (H-bond) networks. In this solid, H+ can be displaced between neighboring water molecules within the H-bond networks and therefore move much faster than in the liquid . This “proton hopping” mechanism may account for the high speed of the lateral diffusion of H+ on the bacterial planar membrane because its nanointerface is expected to harbor highly ordered water molecules, as in the aforementioned data from SFG spectroscopy and FM-AFM [27, 28]. Further studies will be needed to validate this hypothesis.
Functional importance of water molecules in the nanointerface has been ascribed to the gating of K+ channels, as reported by Syeda et al. . In that study, the pore structure without the voltage sensor was reconstituted in lipid bilayers, and the channel activity was measured at different compositions of lipids. When phosphoglycerides located on the extracellular leaflet contained glycerol or inositol, the channel conductance was reduced and the opening time was prolonged. The mechanisms underlying these observations were proposed as follows; the presence of glycerol or inositol caused depletion of water from the exit of the ion conductance path in the channel, and this phenomenon prevented K+ from exiting the filter gate from rehydration. In support of this notion, similar effects on channel activity were observed when small amounts of ethylene glycol or mannitol, which are cryoprotectants that modulate water structure by interacting with hydrogen bonds, are added to the bath solution. In any case, modulation of the channel is likely to be related to the neighboring extracellular water molecules present in the nanointerface.
To further study the iMES, bioanalytical technologies must be developed more actively. For example, the unstirred layer should also contain solutes such as hormones and peptides as mentioned above. For monitoring of the dynamics of these bioactive substances, electrochemical approaches may be applicable. For instance, microelectrodes composed of carbon or conductive diamond can measure concentrations of monoamines [30, 31]. Modification of the surface of the materials with particular compounds may allow the electrodes to detect specific substances in the unstirred layer. An important issue in the development of bioimaging and electrochemical probes will be to avoid their interference with the conditions and milieus of the interphase as much as possible. To this end, coating of the probe’s surface with biocompatible phospholipid polymers would reduce nonspecific absorption of proteins and other substances. This modification was used for the aforementioned fluorescent nanoparticles targeting sialic acid residues, thus resulting in improved sensitivity and specificity .
It is also necessary to clarify how the detected physicochemical phenomena in the unstirred layer and nanointerface are regulated by membrane proteins and how they affect cellular and tissue functions. In situ and in vivo assays should be designed to directly examine the relation between events in the interphase and tissue or organ activities. Experimental and theoretical approaches using phospholipid polymer brush membranes and computational models are also needed. In particular, several simulations of macroscopic ionic dynamics based on ion channels and transporters have been recently described [32, 33]; these could be combined with calculations of microscopic ion dynamics in the iMES. Consolidation of all the data will unveil the physiological and pathological significance of the interphase. These endeavors may establish a new interdisciplinary field of research in life sciences.
We would like to thank Editage (http://www.editage.jp) for English language editing. This work is partially supported by AMED-CREST, AMED (to HH).
Compliance with ethical standards
Conflict of interest
None of the authors has any conflict of interest to report.
This article does not contain any studies with human participants or animals performed by any of the authors.
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