Regulation and dysregulation of intracellular calcium (Ca 2+ ) signaling via the inositol 1,4,5-trisphosphate receptor (InsP 3 R) has been linked to many cellular processes and pathological conditions. In the present study, addition of neuronal calcium sensor-1 (NCS-1), a high-affinity, low-capacity, calcium-binding protein, to purified InsP 3 R type 1 (InsP 3 R1) increased the channel activity in both a calcium-dependent and -independent manner. In intact cells, enhanced expression of NCS-1 resulted in increased intracellular calcium release upon stimulation of the phosphoinositide signaling pathway. To determine whether InsP 3 R1/NCS-1 interaction could be functionally relevant in bipolar disorders, conditions in which NCS-1 is highly expressed, we tested the effect of lithium, a salt widely used for treatment of bipolar disorders. Lithium inhibited the enhancing effect of NCS-1 on InsP 3 R1 function, suggesting that InsP 3 R1/NCS-1 interaction is an essential component of the pathomechanism of bipolar disorder.

In an attempt to identify proteins that assemble with the apical membrane Na ؉ -H ؉ exchanger isoform NHE3, we generated monoclonal antibodies (mAbs) against affinity-purified NHE3 protein complexes isolated from solubilized renal microvillus membrane vesicles. Hybridomas were selected based on their ability to immunoprecipitate NHE3. We have characterized in detail one of the mAbs (1D11) that specifically co-precipitated NHE3 but not villin or NaPi-2. Western blot analyses of microvillus membranes and immunoelectron microscopy of kidney sections showed that mAb 1D11 recognizes a 110-kDa protein highly expressed on the apical membrane of proximal tubule cells. Immunoaffinity chromatography was used to isolate the antigen against which mAb 1D11 is directed. N-terminal sequencing of the purified protein identified it as dipeptidyl peptidase IV (DPPIV) (EC 3.4.14.15), which was confirmed by assays of DPPIV enzyme activity. We also evaluated the distribution of the NHE3-DPPIV complex in microdomains of rabbit renal brush border. In contrast to the previously described NHE3-megalin complex, which principally resides in a dense membrane population (coated pits) in which NHE3 is inactive, the NHE3-DP-PIV complex was predominantly in the microvillar fraction in which NHE3 is active. Serial precipitation experiments confirmed that anti-megalin and anti-DPPIV antibodies co-precipitate different pools of NHE3. Taken together, these studies revealed an unexpected association of the brush border Na ؉ -H ؉ exchanger NHE3 with dipeptidyl peptidase IV in the proximal tubule. These findings raise the possibility that association with DP-PIV may affect NHE3 surface expression and/or activity.The majority of NaCl, NaHCO 3 and water filtered by the kidney is reabsorbed in the proximal tubule. Na ϩ -H ϩ exchange is the predominant mechanism for absorption of Na ϩ and secretion of H ϩ across the apical membrane of proximal tubule cells (1). Several lines of evidence indicate that NHE3 is the Na ϩ -H ϩ exchanger isoform responsible for most, if not all, apical membrane Na ϩ -H ϩ exchange activity in this segment of the nephron (2-7). This isoform thereby plays an important role in the maintenance of fluid and electrolyte balance, and its activity is regulated in response to a wide variety of acute and chronic physiologic stimuli (8 -11).The polarized expression and regulation of a transporter such as NHE3 necessarily involves interactions with other proteins. Recent studies have indicated that NHE3 is capable of binding calmodulin (12), the NHE 1 regulatory factor (NHERF) (11, 13) and its homologue, exchanger-3 kinase A regulatory protein (E3KARP) (14), and the calcineurin B homologous protein (15). These interactions have generally been characterized in nonepithelial cells transfected to overexpress NHE3.We have been investigating whether NHE3 exists in assemblies with other proteins in native kidney membranes. We previously reported that the sedimentation coefficient for NHE3 solubilized from renal membranes is greater than predicte...

We investigated whether the renal brush border Na ؉ /H ؉ exchanger NHE3 exists in assemblies with other proteins in native kidney membranes. To this end we generated monoclonal antibodies (mAbs) against affinity purified NHE3 protein complexes. Hybridomas were selected based on ability to immunoprecipitate NHE3. One of the resulting mAbs (10A3) labeled a high molecular mass (>200 kDa) protein and stained primarily the coated pit region of the proximal tubule in a manner similar to that described for megalin (gp330). We then confirmed that both mAb 10A3 and a known anti-megalin mAb immunoprecipitated and immunoblotted the same protein, namely megalin. mAb 10A3 specifically co-precipitated NHE3 but not villin or NaP i -2 from solubilized renal membranes, indicating specificity of the NHE3-megalin interaction. When immunoprecipitations were performed using either 10A3 or anti-NHE3 mAb 2B9 after separation of solubilized renal proteins by sucrose velocity gradient centrifugation, we found that NHE3 exists in two states with distinct sedimentation coefficients, a 9.6 S megalin-free form and a 21 S megalin-bound form, and that when NHE3 assembles with megalin, epitopes within the carboxyl-terminal 131 amino acids of NHE3 are blocked. Taken together, these findings indicate that a significant pool of NHE3 exists as a multimeric complex with megalin in the brush border of the proximal tubule.

We have previously shown that Na ؉ -H ؉ exchanger isoform NHE3 exists as both 9.6 and 21 S (megalin-associated) oligomers in the renal brush border (1). To characterize the oligomeric forms of the renal brush border Na ؉ -H ؉ exchanger in more detail, we performed membrane fractionation studies. We found that similar amounts of NHE3 were present in microvilli and a nonmicrovillar membrane domain of high density (dense vesicles). Horseradish peroxidase-labeled endosomes were not prevalent in the dense membrane fraction. However, megalin, which localizes primarily to the intermicrovillar microdomain of the brush border, was enriched in the dense vesicles, implicating this microdomain as the likely source of these membranes. Immunolocalization of NHE3 confirmed that a major fraction of the transporter colocalized with megalin in the intermicrovillar region of the brush border. Immunoprecipitation studies demonstrated that in microvilli the majority of NHE3 was not bound to megalin, while in the dense vesicles most of the NHE3 coprecipitated with megalin. Moreover, sucrose velocity gradient centrifugation experiments revealed that most NHE3 in microvilli sedimented with an S value of 9.6, while the S value of NHE3 in dense vesicles was 21. Finally, we examined the functional state of NHE3 in both membrane fractions. As assayed by changes in acridine orange fluorescence, imposing an outwardly directed Na ؉ gradient caused generation of an inside acid pH gradient in the microvilli, indicating Na ؉ -H ؉ exchange activity, but not in the dense vesicles. Taken together, these data demonstrate that renal brush border NHE3 exists in two oligomeric states: a 9.6 S active form present in microvilli and a 21 S, megalin-associated, inactive form in the intermicrovillar microdomain of the apical plasma membrane. Thus, regulation of renal brush border Na ؉ -H ؉ exchange activity may be mediated by shifting the distribution between these forms of NHE3.In the kidney, the activity of the Na ϩ -H ϩ exchanger isoform NHE3, located on the apical microvillar membrane of the proximal tubule, plays a major role in mediating transepithelial bicarbonate and NaCl reabsorption (2-4). Numerous physiologic studies of brush border Na ϩ -H ϩ exchange have shown that this activity is regulated by such hormones as angiotensin II (5) and parathyroid hormone (6) as well as by systemic alterations in acid-base balance (2, 3). Several laboratories have presented evidence suggesting that NHE3 is regulated by posttranslational mechanisms that may include membrane trafficking between an intracellular compartment and the plasma membrane (7-9). Such models predict that NHE3 must be localized in a nonmicrovillar membrane compartment, which functions as a store of transporter, as well as on the microvillar membrane where NHE3 is active.We have recently reported an association between NHE3 and the putative scavenger receptor megalin in renal brush border membranes (1). Moreover, we found that renal brush border NHE3 exists in two states with distinct sediment...

Ca2ϩ signals in neurons use specific temporal and spatial patterns to encode unambiguous information about crucial cellular functions. To understand the molecular basis for initiation and propagation of inositol 1,4,5-trisphosphate (InsP 3 )-mediated intracellular Ca 2ϩ signals, we correlated the subcellular distribution of components of the InsP 3 pathway with measurements of agonist-induced intracellular Ca 2ϩ transients in cultured rat hippocampal neurons and pheochromocytoma cells. We found specialized domains with high levels of phosphatidylinositol-4-phosphate kinase (PIPKI␥) and chromogranin B (CGB), proteins acting synergistically to increase InsP 3 receptor (InsP 3 R) activity and sensitivity. In contrast, Ca 2ϩ pumps in the plasma membrane (PMCA) and sarco-endoplasmic reticulum as well as buffers that antagonize the rise in intracellular Ca 2ϩ were distributed uniformly. By pharmacologically blocking phosphatidylinositol-4-kinase and PIPKI␥ or disrupting the CGB-InsP 3 R interaction by transfecting an interfering polypeptide fragment, we produced major changes in the initiation site and kinetics of the Ca 2ϩ signal. This study shows that a limited number of proteins can reassemble to form unique, spatially restricted signaling domains to generate distinctive signals in different regions of the same neuron. The finding that the subcellular location of initiation sites and protein microdomains was cell type specific will help to establish differences in spatiotemporal Ca 2ϩ signaling in different types of neurons.