Michael Kreman

A method for determining the unitary functional capacity of cloned channels and transporters expressed in Xenopus laevis oocytes

The Xenopus laevis oocyte is widely used to express exogenous channels and transporters and is well suited for functional measurements including currents, electrolyte and nonelectrolyte fluxes, water permeability and even enzymatic activity. It is difficult, however, to transform functional measurements recorded in whole oocytes into the capacity of a single channel or transporter because their number often cannot be estimated accurately. We describe here a method of estimating the number of exogenously expressed channels and transporters inserted in the plasma membrane of oocytes. The method is based on the facts that the P (protoplasmic) face in water-injected control oocytes exhibit an extremely low density of endogenous particles (212±48 particles/μm2, mean, sd) and that exogenously expressed channels and transporters increased the density of particles (up to 5,000/μm2) only on the P face. The utility and generality of the method were demonstrated by estimating the “gating charge” per particle of the Na+/ glucose cotransporter (SGLT1) and a nonconducting mutant of the Shaker K+ channel proteins, and the single molecule water permeability of CHIP (Channel-like Intramembrane Protein) and MIP (Major Intrinsic Protein). We estimated a “gating charge” of ∼3.5 electronic charges for SGLT1 and ∼9 for the mutant Shaker K+ channel from the ratio of Qmax to density of particles measured on the same oocytes. The “gating charges” were 3-fold larger than the “effective valences” calculated by fitting a Boltzmann equation to the same charge transfer data suggesting that the charge movement in the channel and cotransporter occur in several steps. Single molecule water permeabilities (pfs) of 1.4 × 10−14 cm3/ sec for CHIP and of 1.5 × 10−16 cm3/sec for MIP were estimated from the ratio of the whole-oocyte water permeability (Pf) to the density of particles. Therefore, MIP is a water transporter in oocytes, albeit ∼100-fold less effective than CHIP.

Comparison of the Water Transporting Properties of MIP and AQP1

AbstractIn this paper we compare the water-transport properties of Aquaporin (AQP1), a known water channel, and those of the 28 kD Major Intrinsic Protein of Lens (MIP), a protein with an undefined physiological role. To make the comparison as direct as possible we measured functional properties in Xenopus laevis oocytes injected with cRNAs coding for the appropriate protein. We measured the osmotic permeability, Pf, (using rate of swelling) and the surface density of plasma membrane proteins (using freeze-fracture electron microscopy) in the same oocytes. Knowing both Pf and the number of exogenously expressed proteins in the membrane, we estimated the single-molecule permeability to be 2.8 × 10−16 cm3/sec for MIP and 1.2 × 10−14 cm3/sec for AQP1. As a negative control, a mutant MIP, truncated at the carboxyl-terminal, was shown by western blotting to be expressed, but this protein resulted in no increase in either water permeability or particle density. (Interestingly, the truncated protein was glycosylated, while the complete MIP transcript was not.) Water transport by MIP had a higher activation energy (∼7 Kcal/mole) than water transport by AQP1 (∼2.5 Kcal/Mole) but a substantially lower activation energy than water flux across bare oolemma (∼20 Kcal/mole). Though the water-transport properties of MIP and AQP1 differ quantitatively, they are qualitatively quite similar. We conclude that MIP, like AQP1, forms water channels when expressed in oocytes. Thus water transport in the lens seems a plausible physiological role for MIP.

Number of subunits comprising the epithelial sodium channel.

The human epithelial sodium channel (hENaC) is a hetero-oligomeric complex composed of three subunits, α, β, and γ. Understanding the structure and function of this channel and its abnormal behavior in disease requires knowledge of the number of subunits that comprise the channel complex. We used freeze-fracture electron microscopy and electrophysiological methods to evaluate the number of subunits in the ENaC complex expressed in Xenopus laevis oocytes. In oocytes expressing wild-type hENaC (α, β, and γ subunits), clusters of particles appeared in the protoplasmic face of the plasma membrane. The total number of particles in the clusters was consistent with the whole-cell amiloride-sensitive current measured in the same cells. The size frequency histogram for the particles in the clusters suggested the presence of an integral membrane protein complex composed of 17 ± 2 transmembrane α-helices. Because each ENaC subunit has two putative transmembrane helices, these data suggest that in the oocyte plasma membrane, the ENaC complex is composed of eight or nine subunits. At high magnification, individual ENaC particles exhibited a near-square geometry. Functional studies using wild-type αβ-hENaC coexpressed with γ-hENaC mutants, which rendered the functional channel differentially sensitive to methanethiosulfonate reagents and cadmium, suggested that the functional channel complex contains more than one γ subunit. These data suggest that functional ENaC consists of eight or nine subunits of which a minimum of two are γ subunits.

Molecular characterization of Vibrio parahaemolyticus vSGLT: a model for sodium-coupled sugar cotransporters.

The Na+/galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni2+ affinity chromatography and gel filtration. Resequencing the vSGLT gene identified an important correction: the N terminus constitutes an additional 13 functionally essential residues. The mass of His-tagged vSGLT expressed under its native promoter, as determined by electrospray ionization-mass spectrometry (ESI-MS), verifies these 13 residues in wild-type vSGLT. A fusion protein of vSGLT and green fluorescent protein, comprising a mass of over 90 kDa, was also successfully analyzed by ESI-MS. Reconstitution of purified vSGLT yields proteoliposomes active in Na+-dependent galactose uptake, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 stoichiometry and apparent K m values of 129 mm (Na+) and 158 μm(galactose). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monomer. We conclude that vSGLT is a suitable model for the study of sugar cotransporter mechanisms and structure, with potential applicability to the larger SGLT family of important sodium:solute cotransporters. It is further demonstrated that ESI-MS is a powerful tool for the study of proteomics of membrane transporters.

Compound missense mutations in the sodium/D-glucose cotransporter result in trafficking defects

BACKGROUND & AIMS Defects in the Na+-dependent glucose transporter (SGLT1) are associated with the disorder glucose-galactose malabsorption, characterized by severe diarrhea. This study focused on a unique proband with glucose-galactose malabsorption who was investigated 30 years ago, and the aims of the study were to identify mutations in the SGLT1 gene and to determine the defect in sugar transport. METHODS Mutations were identified by sequencing, and each mutant protein was then studied using a Xenopus oocyte heterologous expression system. Analysis included Western, freeze fracture, radiotracer uptake, and electrophysiological assays. RESULTS Two heterozygous missense mutations (Cys355Ser and Leu147Arg) were identified that entirely eliminated Na+/sugar cotransport activity. Western blot analysis showed that the levels of both mutant proteins in the oocyte were comparable to wild-type SGLT1, but no complex glycosylation was detected. No SGLT1 charge movements were observed with the mutant proteins, and freeze fracture data showed that neither mutant protein reached the plasma membrane. CONCLUSIONS The Cys355Ser and Leu147Arg mutations eliminate the Na+/sugar cotransport by blocking the transfer of SGLT1 protein from the endoplasmic reticulum to the plasma membrane. This is consistent with earlier studies on phlorizin binding to the brush border membrane of duodenal biopsy specimens from this patient.

Molecular structure of outer segment disks in photoreceptor cells

The structure of the photoreceptor cell outer segment disks in guinea pig, frog, and gecko retinas were analyzed by means of freeze-fracturing and showed basically identical features. Thanks to a rather accurate locating of the fracture planes based on observations, the observed structure could be interpreted to reveal that the outer segment disk membranes consist of two layers with greatly different physical properties and with each layer measuring about 50 A in thickness. These observations were interpreted on the basis of information obtained earlier by means of polarization optical and x-ray diffraction analysis as well as of what is known about the chemical composition of the photoreceptor cell outer segments. On this basis it was concluded that each disk membrane consists of one lipid bilayer and one monolayer of photopigment molecules dispersed in an aqueous medium of small volume. A particulate structure at the edge of the disk made this part of the disk appear as structurally different from the disk membrane. A hypothetical mechanism is proposed for the transmission of a signal linking energy transduction to the change in permeability of the outer segment plasma membrane caused by absorption of light. The outer segment disk membrane differs structurally extensively from the inner mitochondrial membranes, and the differences were interpreted to reflect the very different functions at a molecular level of these two types of membranes.

Freeze-fracture analysis of structure of plasma membrane of photoreceptor cell outer segments

The plasma membrane of the outer segments of photoreceptor cells in the guinea pig retina was analyzed by means of freeze-fracturing. Two fracture faces with greatly different structure were observed. The relationship of these two faces to the edge which was exposed when the fracture passed across the membrane made it possible to establish that they revealed the cytoplasmic and the peripheral surfaces of the plasma membrane. The thickness of the plasma membrane was measured to be about 100 A. The difference in appearance of the cytoplasmic and the peripheral surfaces of the plasma membrane was interpreted to reveal a layered structure with a predominantly lipid layer at the cytoplasmic surface, and a predominantly protein layer at the peripheral surface. A concept describing the molecular structure of the membrane is proposed. The outer segment plasma membrane differs structurally from the outer segment disk membranes. Certain aspects of disk membrane formation as invaginations of the plasma membrane are discussed. The fact that only two fracture faces were observed and no complementary faces was explained by a simple analysis of some effects of the fracturing on the frozen material.

Freeze-fracture analysis of photoreceptor cell outer segment disks after minimal extraction of rhodopsin.

The outer segment disk membranes in the photoreceptor cells of the guinea pig retinas were analyzed after partial extraction of rhodopsin with cetyltrimethylammonium bromide (CTAB). During the earliest stages of rhodopsin extraction, the particulate structure of the particulate face of the disk membranes becomes less dense with the appearance of smooth areas extending between the particles. These smooth areas could occupy a large part of the particulate membrane face. The disk membranes could still retain a planar structure. A transformation of the disk membranes into small vesicles was also evident and was considered to correspond to a stage where the extraction has progressed beyond the earliest stages. Extraction with digitonin led to an immediate disintegration of the disk membranes into vesicles. Since CTAB can preferentially solubilize rhodopsin without extracting the membrane lipids ( Heller, 1968; Biochemistry , 7 , 2906 ), the observations were considered to support the interpretation of Sjostrand and Kreman (1978, J. Ultrastruct. Res. , 65 , 195) , that the surface structure of the particulate membrane face is due predominantly to rhodopsin molecules forming a separate layer in the disk membrane.

Intercellular fibrillar skeleton in the basal interdigitations of kidney tubular cells

SummaryThe tubular cells from the thick ascending limb of the loop of Henle in rabbit kidney medulla contain in their basallateral surfaces a complex system of interdigitations. Within these interdigitations, the plasma membranes are separated by extracellular spaces of relatively constant width that contain a previously undescribed fibrillar system. The structural organization and distribution of this intercellular fibrillar skeleton was studied using freeze-fracture etch and then section electron microscopy. The skeleton is comprised of discrete strands with a density of 300 to 400 per μm2 evenly distributed along the entire basal-lateral region. Each strand has the shape of a brace and it is constructed from up to eight finer filaments each having a width of about 2 nm. The filaments are tightly joined together along their shafts for about 30 nm but they separate at both ends for about 10 nm before contacting the external surface of the plasma membrane. We propose that this intercellular fibrillar skeleton is responsible for maintaining the wide (about 50 nm) and uniform plasma membrane separation along the entire length of the basallateral region of the tubular cells of the thick ascending limb.