Observation of enhanced transmission for s-polarized light through a subwavelength slit.

Enhanced optical transmission (EOT) through a single aperture is usually achieved by exciting surface plasmon polaritons with periodic grooves. Surface plasmon polaritons are only excited by p-polarized incident light, i.e. with the electric field perpendicular to the direction of the grooves. The present study experimentally investigates EOT for s-polarized light. A subwavelength slit surrounded on each side by periodic grooves has been fabricated in a gold film and covered by a thin dielectric layer. The excitation of s-polarized dielectric waveguide modes inside the dielectric film strongly increases the s-polarized transmission. A 25 fold increase is measured as compared to the case without the dielectric film. Transmission measurements are compared with a coupled mode method and show good qualitative agreement. Adding a waveguide can improve light transmission through subwavelength apertures, as both s and p-polarization can be efficiently transmitted.

Self-collimating photonic crystal polarization beam splitter.

We present theoretical and experimental results of a polarization splitter device that consists of a photonic crystal (PhC) slab, which exhibits a large reflection coefficient for TE and a high transmission coefficient for TM polarization. The slab is embedded in a PhC tile operating in the self-collimation mode. Embedding the polarization-discriminating slab in a PhC with identical lattice symmetry suppresses the in-plane diffraction losses at the PhC-non-PhC interface. The optimization of the PhC-non-PhC interface is thereby decoupled from the optimization of the polarizing function. Transmissions as high as 35% for TM- and 30% for TE-polarized light are reported.

Planar photonic crystals infiltrated with liquid crystals: optical characterization of molecule orientation.

Nematic liquid crystals are infiltrated into InP-based planar photonic crystals. Optical measurements as a function of temperature and polarization are used to study the average director field configuration in the nanometer-size holes: a planar equilibrium state is found.

Ion pumps in polarized cells: sorting and regulation of the Na+, K+- and H+, K+-ATPases.

The physiologic function of an ion transport protein is determined, in part, by its subcellular localization and by the cellular mechanisms that modulate its activity. The Na(+),K(+)-ATPase and the H(+),K(+)-ATPases are closely related members of the P-type family of ion transporting ATPases. Despite their homology, these pumps are sorted to different domains in polarized epithelial cells, and their enzymatic activities are subject to distinct regulatory pathways. The molecular signals responsible for these properties have begun to be elucidated. It appears that a complex array of inter- and intramolecular interactions govern trafficking, distribution, and catalytic capacities of these proteins.

The cell biology of ion pumps: sorting and regulation.

The physiologic function of an ion pump is determined, in part, by its subcellular localization and by the cellular mechanisms that modulate its activity. The Na,K-ATPase and the gastric H,K-ATPase are two closely related members of the P-type family of ion transporting ATPases. Despite their homology, these pumps are sorted to different domains in polarized epithelial cells and their enzymatic activities are subject to distinct regulatory pathways. The molecular signals responsible for these properties have begun to be elucidated. It appears that a complex array of inter- and intra-molecular interactions govern these proteins' trafficking, distribution and catalytic capacity.

A transmembrane segment determines the steady-state localization of an ion-transporting adenosine triphosphatase.

The H,K-adenosine triphosphatase (ATPase) of gastric parietal cells is targeted to a regulated membrane compartment that fuses with the apical plasma membrane in response to secretagogue stimulation. Previous work has demonstrated that the alpha subunit of the H, K-ATPase encodes localization information responsible for this pump's apical distribution, whereas the beta subunit carries the signal responsible for the cessation of acid secretion through the retrieval of the pump from the surface to the regulated intracellular compartment. By analyzing the sorting behaviors of a number of chimeric pumps composed of complementary portions of the H, K-ATPase alpha subunit and the highly homologous Na,K-ATPase alpha subunit, we have identified a portion of the gastric H,K-ATPase, which is sufficient to redirect the normally basolateral Na,K-ATPase to the apical surface in transfected epithelial cells. This motif resides within the fourth of the H,K-ATPase alpha subunit's ten predicted transmembrane domains. Although interactions with glycosphingolipid-rich membrane domains have been proposed to play an important role in the targeting of several apical membrane proteins, the apically located chimeras are not found in detergent-insoluble complexes, which are typically enriched in glycosphingolipids. Furthermore, a chimera incorporating the Na, K-ATPase alpha subunit fourth transmembrane domain is apically targeted when both of its flanking sequences derive from H,K-ATPase sequence. These results provide the identification of a defined apical localization signal in a polytopic membrane transport protein, and suggest that this signal functions through conformational interactions between the fourth transmembrane spanning segment and its surrounding sequence domains.

Residues of the fourth transmembrane segments of the Na,K-ATPase and the gastric H,K-ATPase contribute to cation selectivity.

We have generated protein chimeras to investigate the role of the fourth transmembrane segments (TM4) of the Na,K- and gastric H, K-ATPases in determining the distinct cation selectivities of these two pumps. Based on a helical wheel analysis, three residues of TM4 of the Na,K-ATPase were changed to their H,K-counterparts. A construct carrying three mutations in TM4 (L319F, N326Y, and T340S) and two control constructs were heterologously expressed in Xenopus laevis oocytes and in the pig kidney epithelial cell line LLC-PK(1). Biochemical ATPase assays demonstrated a large sodium-independent ATPase activity at pH 6.0 for the pump carrying the TM4 substitutions, whereas the control constructs exhibited little or no activity in the absence of sodium. Furthermore, at pH 6.0 the K(1/2)(Na(+)) shifted to 1.5 mM for the TM4 construct compared with 9.4 and 5.9 mM for the controls. In contrast, at pH 7.5 all three constructs had characteristics similar to wild type Na,K-ATPase. Large increases in K(1/2)(K(+)) were observed for the TM4 construct compared with the control constructs both in two-electrode voltage clamp experiments in Xenopus oocytes and in ATPase assays. ATPase assays also revealed a 10-fold shift in vanadate sensitivity for the TM4 construct. Based on these findings, it appears that the three identified TM4 residues play an important role in determining both the specific cation selectivities and the E(1)/E(2) conformational equilibria of the Na,K- and H,K-ATPase.

Nongastric H+,K+-ATPase: cell biologic and functional properties.

Several members of the H+,K+-ATPase family of ion pumps participate in renal K transport. This class of P-type ATPases includes the gastric H+,K+-ATPase as well as a number of nongastric H+,K+-ATPase isoforms. Physiological studies suggest that these enzymes operate predominantly at the apical surfaces of tubule epithelial cells. Although much has been learned about the pattern of H+,K+-ATPase isoform expression and its response to stress, the functional and cell biologic attributes of these pumps remain largely unelucidated. We have studied the properties of renal H+,K+-ATPases both in vitro and in situ. Our analysis of ion fluxes driven by a nongastric H+,K+-ATPase isoform suggests that it exchanges Na (rather than H) for K under normal circumstances. Thus, the individual H+,K+-ATPase isoforms may make diverse contributions to renal cation transport. We find that the activities of renal H+,K+-ATPases in situ are regulated by endocytosis, which is mediated by an endocytosis signal in the cytoplasmic tail of the gastric H+,K+-ATPase beta-subunit. Transgenic mice expressing a version of this protein in which the signal has been disabled show constitutively active renal K resorption. The identities of the H+,K+-ATPase isoforms that are normally subject to endocytic regulation and the nature of the participating epithelial cell machinery have yet to be established.

Sorting of P-type ATPases in polarized epithelial cells.

The Na,K-ATPase and the H,K-ATPase are highly homologous members of the P-type family of ion transporting ATPase. Despite their structural similarity, these two pumps are sorted to different destinations in polarized epithelial cells. While the Na,K-ATPase is restricted to the basolateral surfaces of most epithelial cells types, the H,K-ATPase is concentrated at the apical plasmalemma and in a pre-apical vesicular storage compartment in the parietal cells of the stomach. We have generated molecular chimeras composed of complementary portions of these two pumps' alpha-subunits. By expressing these pump constructs in polarized epithelial cells in culture, we have been able to identify sequence domains which participate in the targetting of the holoenzyme. We find that information embedded within the sequence of the fourth transmembrane domain of the H,K-ATPase is sufficient to account for this protein's apical localization. Stimulation of gastric acid secretion results in insertion of the intracellular H,K-ATPase pool into the apical plasma membrane and inactivation of acid secretion is accompanied by the re-internalization of these pumps. We have identified a tyrosine-based signal in the cytoplasmic tail of the H,K-ATPase beta-subunit which appears to be required for this endocytosis. We have mutated the critical tyrosine residue to alanine and expressed the altered protein in transgenic mice. The H,K-ATPase remains continuously at the apical cell surface in parietal cells from these animals, and they constitutively hypersecrete gastric acid. These results demonstrate that the beta-subunit sequence mediates the internalization of the H,K-ATPase and is required for the cessation of gastric acid secretion. Thus, at least two sorting signals are required to ensure the proper targetting and regulation of the gastric H,K pump.

Sorting and trafficking of ion transport proteins in polarized epithelial cells.

Transport proteins are targeted to specific plasmalemmal domains in polarized epithelial cells. The molecular signals that govern these sorting events are just beginning to be elucidated. In many cases, the cell surface delivery of transport proteins is subjected to tight regulation. Several different mechanisms appear to participate in these trafficking processes.

Biotinylation and assessment of membrane polarity: caveats and methodological concerns.

Studies of epithelial membrane polarity have been greatly facilitated through the use of the N-hydroxysuccinimide-biotin surface labeling technique (M. Sargiacomo, M. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. J. Membr. Biol. 107: 277-286, 1989). We have used this technique in studies on the sorting and targeting of ion-transporting adenosinetriphosphatase molecules in polarized epithelial cells. Through efforts to optimize this technique in our experimental system, we have encountered several experimental conditions and circumstances where biotinylation is extremely inefficient and the assessment of membrane polarity which it provides is misleading. We demonstrate that the pH and ionic strength of the biotinylation buffer can dramatically affect biotin incorporation and that protocol-dependent variations in the recovery of biotinylated proteins can result in misrepresentation of the actual apical/basolateral distribution of a protein. Conditions and protocols that may improve the sensitivity and accuracy of this technique are discussed.