John Cuppoletti

Cytochalasin b binding to ehrlich ascites tumor cells and its relationship to glucose carrier.

Cultured Ehrlich ascites tumor cells equilibrate D-glucose via a carrier mechanism with a Km and V of 14 mM and 3 mu mol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (Ki) of approx. 5.10(-7) M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1.10(-5) M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (Kd) of approx. 1.10(-6) M, represents about 30% of the total cytochalasin B binding of the cell (8.10(6) molecules/cell), is sensitively displaced by cytochalasin E but not by D-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a Kd of 4--6.10(-7) M, represents approx. 60% of the total saturable binding (14.10(6) molecules/cell), is specifically displaced by D-glucose with a displacement constant of 15 mM, but not by L-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a Kd of 2--6.10(-8) M, represents less than 10% of the total sites (2.10(6) molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.

Gastric H+ secretion: histamine (cAMP-mediated) activation of protein phosphorylation

Activation of H+ secretion by the gastric parietal cell involves major changes in morphology, metabolic activity and ion pathways of the secretory membrane. These changes are elicited by histamine binding to the H2 receptor, raising cAMP levels and presumably activating cAMP-dependent protein kinase. Concomitantly, the intracellular free Ca2+ concentration, [Ca2+]i, increases. Studies were performed to determine whether cAMP-mediated protein phosphorylation accompanies histamine activation of H+ secretion and to catalogue the major protein species serving as substrates for cAMP-dependent protein kinase in the parietal cell. 80% pure rabbit parietal cells, prepared by Nycodenz bouyant density centrifugation, were used. To investigate only cAMP-mediated effects, histamine-dependent changes in [Ca2+]i in these cells were abolished by depleting intracellular Ca2+ stores and performing experiments under Ca2+-free conditions. Acid secretion and steady-state levels of protein phosphorylation were then measured in unstimulated (cimetidine-treated) and histamine-stimulated cells. In intact parietal cells, concommitant with histamine stimulation of H+ secretion, increases in the level of protein phosphorylation were observed. Significantly changing phosphoproteins found in supernatant fractions showed apparent subunit sizes of approx. 148, 130, 47 and 43 kDa, and in microsomal fractions included those at approx. 130, 51 and 47 kDa. In parietal cell homogenates, using [gamma-32P]ATP, cAMP elicited significant phosphorylation of eight supernatant proteins and twelve microsomal proteins, which included the histamine-dependent phosphoproteins found in the intact parietal cell, except for the 51 kDa microsomal protein. As a working hypothesis, these proteins are involved in stimulus-secretion coupling in the parietal cell.

Kir2.1 K+ Channels of the Gastric Parietal Cell

The purpose of this article is to review the literature regarding the characteristics of the gastric parietal cell apical membrane K+ channel and provide new evidence that suggests that the inward rectifying K+ channel, Kir2.1, may be involved in K+ recycling at the apical membrane of the gastric parietal cell. It has been previously reported that gastric H+/K+ ATPase-containing vesicles exhibit a 10 pS K+ channel when measured in planar lipid bilayers. The native gastric K+ channel in these vesicles is insensitive to ATP, is active in the absence of Ca2+, is stable at an extracellular pH of 3.0, and is activated by PKA. A literature search suggested that Kir2.1 has many of the properties of this channel. This is the first report to show that recombinant rabbit Kir2.1 K+ channels are active in the presence of ATP, in the absence of Ca2+, and are stable at an extracellular pH of 3.0. Preliminary results demonstrate that Kir2.1 is also PKA activated. Kir2.1 also exhibits single channel currents that are of similar magnitude as the native channel under these conditions. In an accompanying paper, Grahammer et al suggest from 293 B inhibition of acid secretion that KCNQ1 is the K+ channel involved in K+ recycling at the apical membrane. Kir2.1 is not inhibited by 293B. Acid accumulation by H+/K+ ATPase-containing vesicles from stimulated rabbits is insensitive to 293B. This finding strongly suggests that KCNQ1 is not involved in K+ recycling at the apical membrane of the gastric parietal cell. Kir2.1 is a candidate for this process.

Ion Pumps, Ion Pathways, Ion Sites

There has been a rapidly growing list of ATP-fueled ion pumps. Most of these are H+-transporting ATPases ranging from the mitochondrial F1F0 ATPase through the H+ pump of intracellular organelles to the gastric H+-K+ ATPase (Racker, 1976; Cidon and Nelson, 1983; Sachs et al, 1976). In addition to the Ca2+ ATPase of sarcoplasmic reticulum and plasma membrane (Schatzman, 1982), the role of the Ca2+ pump of rough endoplasmic reticulum in fueling a facultative Ca2+ store has been recognized (Berridge, 1984), and its properties are currently of interest.

Engineered Membranes and Transporters for Useful Devices

Biological membranes consist of lipid bilayers which are permeant to gases and water and some hydrophobic substances, but are impermeant to charged materials such as ions. Biological membranes in cells contain a variety of proteins (ion transporters, ion channels, pumps and other types of pore proteins) to facilitate the transport of ions and other molecules such as glucose and even large molecules such as proteins. The present chapter describes our studies to form bilayers on artificial porous supports and solid supports, and incorporate transporters and ion channels into such artificial membranes in functional form. We were then able to remove ion channels from cells, and place them onto a wide variety of solid and permeant synthetic supports without loss of function. While the above studies involved native ion transport proteins, we also have undertaken studies to modify ion channels to give useful properties. Starting with ion channels of known sequence and crystal structures, these studies outlined the structural basis for functional and regulatory properties. The approaches taken to prepare the engineered composite membranes are generally applicable to the development, design and prediction of properties of a wide variety of materials such as selectively permeable membranes or functionalized thin films with desired chemical, electrical or optical properties. ClC-2 Cltransporting channels, potassium ion channels, and transporters were used for this work. The following major developments have facilitated this phase of the work. First, the primary structure of the ion channels was known from cloning. Second, the X-ray crystal structure and additional NMR structures of a ClC Clchannel have been published. Our group was been able to use both the primary structure and the structural information to develop experiments to engineer new properties into the channels. Recent NMR and X-ray crystal structural studies have given important new information regarding the structure of the intracellular C-terminal region of ClC-2, and this information helped to explain the structural basis for our findings that this same region is involved in phosphorylation-dependent regulation of the channel. Dissection and reconstitution of this region has already been carried out, raising our level of confidence that we can exploit this regulatory region further in future studies, such as