The Rh blood group proteins are well known as the erythrocyte targets of the potent antibody response that causes hemolytic disease of the newborn. These proteins have been described in molecular detail; however, little is known about their function. A transport function is suggested by their predicted structure and from phylogenetic analysis. To obtain evidence for a role in solute transport, we expressed Rh proteins in Xenopus oocytes and now demonstrate that the erythroid Rh-associated glycoprotein mediates uptake of ammonium across cell membranes. Rh-associated glycoprotein carrier-mediated uptake, characterized with the radioactive analog of ammonium [ 14 C]methylamine (MA), had an apparent EC 50 of 1.6 mM and a maximum uptake rate (V max ) of 190 pmol/oocyte/min. Uptake was independent of the membrane potential and the Na ؉ gradient. MA transport was stimulated by raising extracellular pH or by lowering intracellular pH, suggesting that uptake was coupled to an outwardly directed H ؉ gradient. MA uptake was insensitive to additions of amiloride, amine-containing compounds tetramethyl-and tetraethylammonium chloride, glutamine, and urea. However, MA uptake was significantly antagonized by ammonium chloride with inhibition kinetics (IC 50 ‫؍‬ 1.14 mM) consistent with the hypothesis that the uptake of MA and ammonium involves a similar H ؉ -coupled counter-transport mechanism.The human Rh blood group proteins have been known for decades to cause hemolytic disease of the newborn, which can result in severe fetal morbidity and mortality (1). Despite their clinical importance, these multipass membrane proteins were not successfully isolated until the late 1980s (2), and little progress was made in their characterization until the genes were cloned (3). Database searches for protein sequences with similarities to Rh proteins were not informative until the Caenorhabditis elegans sequencing initiative revealed that homologs exist in that species. These homologs, in turn, revealed a distant similarity between Rh proteins and ammonium transporters from bacteria and yeast. Since then additional Rh homologs have been found in many organisms (4), and nonerythroid Rh homologs were detected in human and mouse kidney, testis, brain, and liver (5, 6).The erythrocyte Rh blood group antigens are carried on two 417-amino acid polypeptides, RhD and RhCE, which are 97% identical. Rh-negative individuals carry a deletion or mutation in RHD and lack RhD protein (for reviews, see Refs. 7 and 8). A related 409-amino acid Rh-associated glycoprotein (RhAG)

2؉ release has been attributed to the ability of the InsP 3 R channels to cluster and to be localized to discrete areas, suggesting that mechanisms may exist to restrict their movement. Here, we examined the lateral mobility of the type 3 isoform of the InsP 3 R (InsP 3 R3) in the ER membrane by performing confocal fluorescence recovery after photobleaching of an InsP 3 R3 with green fluorescent protein fused to its N terminus. In Chinese hamster ovary and COS-7 cells, the diffusion coefficient D was ϳ4 ؋ 10 ؊10 cm 2 /s at room temperature, a value similar to that determined for other ER-localized integral membrane proteins, with a high fraction (ϳ75%) of channels mobile. D was modestly increased at 37°C, and it as well as the mobile fraction were reversibly reduced by ATP depletion. Although disruption of the actin cytoskeleton (latrunculin) was without effect, disruption of microtubules (nocodazole) reduced D by half without affecting the mobile fraction. We conclude that the entire ER is continuous in these cells, with the large majority of InsP 3 R3 channels free to diffuse throughout it, at rates that are comparable with those measured for other polytopic ER integral membrane proteins. The observed InsP 3 R3 mobility may be higher than its intrinsic diffusional mobility because of additional ATPand microtubule-facilitated motility of the channel.The inositol trisphosphate signaling pathway is present in nearly all cells. Activation of phospholipase C by G proteincoupled receptors and receptor tyrosine kinases results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate to two products, diacylglycerol and the watersoluble inositol 1,4,5-trisphosphate (InsP 3 ) 1 (1). InsP 3 diffuses in the cytoplasm and binds to a receptor, the InsP 3 R, a polytopic integral membrane protein in the endoplasmic reticulum (ER), activating it as a Ca 2ϩ channel to liberate stored Ca 2ϩ from the ER lumen into the cytoplasm. This rapid release of Ca 2ϩ modulates the cytoplasmic free Ca 2ϩ concentration ([Ca 2ϩ ] i ), providing a ubiquitous intracellular signal with complex features that endow it with high temporal and spatial specificity (1, 2). The InsP 3 -mediated [Ca 2ϩ ] i signaling system regulates a diversity of cellular processes, including gene transcription, membrane transport, secretion, contraction, intercellular communication, and synaptic plasticity (1, 3).The InsP 3 signaling pathway is expressed ubiquitously, but it nevertheless provides highly spatially and temporally specific [Ca 2ϩ ] i signals. This has been attributed to the diversity of InsP 3 R isoform expression (a family of three InsP 3 R isoforms exists with different primary sequences derived from different genes with alternatively spliced forms (4, 5)), subcellular distributions of the InsP 3 R channels (6 -10), and complex regulation of the channel by both InsP 3 and [Ca 2ϩ ] i (1,11,12). Gating of the InsP 3 -liganded InsP 3 R channel is modulated with a biphasic dependence on [Ca 2ϩ ] i (13-16). Importantly, Ca 2ϩ bi...

Vasoactive intestinal peptide (VIP) stimulates active Cl- secretion by the intestinal epithelium, a process that depends upon the maintenance of a favorable electrical driving force established by a basolateral membrane K+ conductance. To demonstrate the role of this K- conductance, we measured short-circuit current (I(SC)) across monolayers of the human colonic secretory cell line, T84. The serosal application of VIP (50 nM) increased I(SC) from 3 +/- 0.4 microA/cm2 to 75 +/- 11 microA/cm2 (n = 4), which was reduced to a near zero value by serosal applications of Ba2+ (5 mM). The chromanol, 293B (100 microM), reduced I(SC) by 74%, but charybdotoxin (CTX, 50 nM) had no effect. We used the whole-cell voltage-clamp technique to determine whether the K+ conductance is regulated by cAMP-dependent phosphorylation in isolated cells. VIP (300 nM) activated K+ current (131 +/- 26 pA, n = 15) when membrane potential was held at the Cl- equilibrium potential (E(Cl-) = -2 mV), and activated inward current (179 +/- 28 pA, n = 15) when membrane potential was held at the K+ equilibrium potential (E(K+) = -80 mV); however, when the cAMP-dependent kinase (PKA) inhibitor, PKI (100 nM), was added to patch pipettes, VIP failed to stimulate these currents. Barium (Ba2+ , 5 mM), but not 293B, blocked this K+ conductance in single cells. We used the cell-attached membrane patch under conditions that favor K + current flow to demonstrate the channels that underlie this K+ conductance. VIP activated inwardly rectifying channel currents in this configuration. Additionally, we used fura-2AM to show that VIP does not alter the intracellular Ca2+ concentration, [Ca2 +]i. Caffeine (5 mM), a phosphodiesterase inhibitor, also stimulated K+ current (185 +/- 56 pA, n = 8) without altering [Ca2+]i. These results demonstrate that VIP activates a basolateral membrane K+ conductance in T84 cells that is regulated by cAMP-dependent phosphorylation.