Na؉ /H ؉ exchanger NHE3 is a plasma membrane (PM) protein, which contributes to Na ؉ absorption in the intestine. Growth factors stimulate NHE3 via phosphatidylinositol 3-kinase (PI3-K), but mechanism of this process is not clear. To examine the hypothesis that growth factors stimulate NHE3 by modulating NHE3 recycling, and that PI3-K participates in this mechanism, we used PS120 fibroblasts expressing a fusion protein of NHE3 and green fluorescent protein. At steady state, ϳ25% of cellular NHE3 content was expressed at PM. Inhibition of PI3-K decreased PM expression of NHE3, which correlated with retention of the exchanger in recycling endosomal compartment. In contrast, basic fibroblast growth factor (bFGF) increased PM expression of NHE3, which was associated with a 2-fold increase in rate constant for exit of the exchanger from the recycling compartment. Qualitatively similar effects of bFGF were observed in cells pretreated with PI3-K inhibitors, but their magnitude was only ϳ50% of that in intact cells. These data suggest that: (i) bFGF stimulates NHE3 by increasing PM expression of the exchanger; (ii) PI3-K mediates PM expression of NHE3 in both basal and bFGF-stimulated conditions, and (iii) not all of the effects of bFGF on NHE3 expression are mediated by PI3-K, suggesting additional regulatory mechanisms.In the mammalian intestine, sodium and water are reabsorbed by multiple mechanisms which include the activity of Na ϩ /H ϩ exchanger NHE3. 1 This transmembrane protein is expressed in the epithelium of renal tubules, intestine, gall bladder, and salivary gland, where it was localized to the apical microvillar domain and, at least in the kidney and in the intestine, to an yet undefined cytoplasmic compartment (1-4).In the small intestine, NHE3 participates in neutral NaCl absorption, and in the increase in Na ϩ absorption that occurs via neurohormonal stimulation after meals (5). The activity of NHE3 is acutely regulated by multiple mechanisms involving growth factors and protein kinases (6). We and others have shown that stimulation of NHE3 activity by growth factors, okadaic acid, and serum occurs via an increase in the maximal velocity (V max ) of the exchange, whereas phorbol ester and carbachol inhibits NHE3 via a decrease in V max (6). These effects were observed in non-polarized mesenchymal cells as well as in epithelial cells, and they suggested that at least part of the acute regulation might be accomplished by rapid changes in the number of active exchanger molecules at the plasma membrane.Over the last few years, a growing body of evidence has indicated that NHE3 might, indeed, be regulated by redistribution of the exchanger molecules between the cytoplasm and the plasma membrane. Thus, recycling of NHE3 has been suggested in kidney epithelial cells based on the results of subcellular fractionation experiments (7,8), and on the presence of an intracellular compartment accumulating NHE3 (1). Moreover, the protein kinase C-mediated inhibition of endogenous NHE3 in the human colonic adenocarcino...

The P segments of the voltage-dependent Na+ channel line the outer mouth and selectivity filter of the pore. The residues that form the cytoplasmic mouth of the pore of the channel have not been identified. To study the structure of the inner pore mouth, the presumed selectivity filter residues (D400, E755, K1237, and A1529), and three amino acids just amino-terminal to each of these residues in the rat skeletal muscle Na+ channel, were mutated to cysteine and expressed in tsA 201 cells. These amino acids are predicted (by analogy to K+ channels) to be on the cytoplasmic side of the putative selectivity filter residues. Inward and outward Na+ currents were measured with the whole-cell configuration of the patch-clamp technique. Cysteinyl side-chain accessibility was gauged by sensitivity to Cd2+ block and by reactivity with methanethiosulfonate (MTS) reagents applied to both the inside and the outside of the cell. Outward currents through the wild-type and all of the mutant channels were unaffected by internal Cd2+ (100 microM). Similarly, 1 mM methanethiosulfonate ethylammonium (MTSEA) applied to the inside of the membrane did not affect wild-type or mutant outward currents. However, two mutants amino-terminal to the selectivity position in domain III (F1236C and T1235C) and one in domain IV (S1528C) were blocked with high affinity by external Cd2+. The Na+ current through F1236C and S1528C channels was inhibited by MTSEA applied to the outside of the cell. The accessibility of these mutants to externally applied cysteinyl ligands indicates that the side chains of the mutated residues face outward rather than inward. The K+ channel model of the P segments as protein loops that span the selectivity region is not applicable to the Na+ channel.

The pores of ion channel proteins are often modeled as static structures. In this view, selectivity reflects rigidly constrained backbone orientations. Such a picture is at variance with the generalization that biological proteins are flexible, capable of major internal motions on biologically relevant time scales. We tested for motions in the sodium channel pore by systematically introducing pairs of cysteine residues throughout the pore-lining segments. Two distinct pairs of residues spontaneously formed disulfide bonds bridging domains I and II. Nine other permutations, involving all four domains, were capable of disulfide bonding in the presence of a redox catalyst. The results are inconsistent with a single fixed backbone structure for the pore; instead, the segments that line the permeation pathway appear capable of sizable motions.

S U M M A R YWe developed a confocal morphometric analysis to quantitate the relative plasma membrane (PM) expression of the Na/H exchanger NHE3 in living PS120 fibroblasts. NHE3 is a membrane transport protein that is acutely regulated by changes in the number of molecules expressed at the PM. To quantitate the PM expression of NHE3 under various experimental conditions, we stably expressed a chimera of rabbit NHE3 and green fluorescent protein (NHE3-GFP) in PS120 fibroblasts. A three-dimensional (3D) map of the intracellular distribution of NHE3-GFP was obtained by confocal laser scanning microscopy (CLSM) of cells superfused with a styryl dye, FM 4-64. This fluorophore rapidly and reversibly labeled the outer lipid layer of the PM, which allowed generation of a digital mask of the PM and calculation of the fraction of a total cellular NHE3-GFP expressed at the PM. This analysis was successfully used to quantitate the relative PM expression of NHE3-GFP in control cells (25%) and a decrease in the expression caused by subsequent exposure of cells to wortmannin (5.1%). Reliability of the method was confirmed by cell surface biotinylation, which yielded very similar results. Confocal morphometric analysis is fast and reproducible and could potentially be used for investigations on regulation of expression of other membrane proteins. The sodium / hydrogen exchanger NHE3 is a transmembrane protein expressed at the apical membrane domain of Na ϩ -absorbing epithelia, predominantly in the kidney and in the small and large intestine. The protein performs Na/H exchange with stoichiometry 1:1 and is driven by the Na ϩ concentration gradient across the plasma membrane (reviewed in Donowitz et al. 1996). In the kidney, NHE3 plays a role in net NaCl, HCO 3 , and NH 4 reabsorption in renal tubules (Paillard 1997). In the intestine, the exchanger participates in both basal and neurohormonally induced postprandial NaCl absorption in the intestine (reviewed in Donowitz et al. 1999). Acute regulation of NHE3 activity by protein kinases and growth factors occurs within minutes and is mediated predominantly by changes in maximal velocity (V max ) of the exchange (Donowitz et al. 1999). Such a mechanism suggests rapid changes either in the number of NHE3 molecules at the plasma membrane (PM) or in the turnover number (number of exchange cycles per molecule per second), or both. Recent evidence obtained from studies on non-epithelial and on polarized epithelial cells suggest that rapid changes in NHE3 activity might indeed be mediated by removal and/or insertion of the exchanger molecules from the plasma membrane (D'Souza et

Cardiac ATP-sensitive K+ (KATP) channels (SUR2A plus Kir6.2) couple the metabolic state of the myocyte to its electrical activity via a mechanism that is not well understood. Recent pharmacological evidence suggests that KATP channels may mediate ischemic preconditioning. However, there is no potent pharmaceutical agent that specifically blocks the sarcolemmal KATP channel without significant effects on other cellular proteins. As a molecular tool, the GFG sequence in the H5 loop of the murine Kir6.2 channel was mutated to AFA. This mutated channel subunit (6.2AFA) suppressed wild-type Kir6.2 (6.2WT) channel current in a dominant-negative manner: when co-expressed with SUR2A and 6.2WT, whole-cell KATP current recorded from HEK cells was greatly attenuated. The 6.2AFA subunit also co-assembled with endogenous subunits in both smooth-muscle-derived A10 cells and rat neonatal ventricular myocytes, resulting in a significant reduction of current compared with that recorded from non-transfected or mock-transfected cells (<15% of control for both cell types). This study shows that mutation of GFG-->AFA in the putative pore-forming region of Kir6.2 acts in a dominant-negative manner to suppress current in heterologous systems and in native cells.

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