Two histidine residues in the juxta-membrane cytoplasmic domain of Na+/H+ exchanger isoform 3 (NHE3) determine the set point.

Histidine residues in Na+/H+ exchangers are believed to participate in proton binding and influence the Na+/H+ exchanger activity. In the present study, the function of three highly conserved histidines in the juxtamembrane cytoplasmic domain of NHE3 was studied. His-479, His-485, and His-499 were mutated to Leu, Gln or Asp and expressed in an Na+/H+ exchanger null cell line and functional consequences on Na+/H+ exchange kinetics were characterized. None of the histidines were essential for NHE3 activity, with all mutated NHE3 resulting in functional exchangers. However, the mutation in His-475 and His-499 significantly lowered NHE3 transport activity, whereas the mutation in H485 showed no apparent effect. In addition, the pH profiles of the H479 and H499 mutants were shifted to a more acidic region, and lowered its set point, the intracellular pH value above which the Na+/H+ exchanger becomes inactive, by approximately 0.3-0.6 pH units. The changes in set point by the mutations were further shifted to more acidic values by ATP depletion, indicating that the mechanism by which the mutations on the histidine residues altered the NHE3 set point differs from that caused by ATP depletion. We suggest that His-479 and His-499 are part of the H+ sensor, which is involved in determining the sensitivity to the intracellular H+ concentration and Na+/H+ exchange rate.

Calreticulin functions as a molecular chaperone for the beta-amyloid precursor protein.

Processing of the beta-amyloid precursor protein (APP) in the endoplasmic reticulum and the Golgi apparatus may be critical in generating the beta-amyloid molecules linked to the pathogenesis of Alzheimer's disease. Since chaperone molecules such as calreticulin (Crt) have been shown to be important in the maturation of many glycoproteins, we investigated the interaction between Crt and APP. We show that APP binds transiently to Crt in a manner that is pH, divalent cation, and N-linked glycosylation-dependent. Both immature APP (containing only N-linked sugars) and mature APP (containing both N-linked and O-linked sugars) bind to Crt. Both proteins are part of a complex that appears to be large enough to accommodate other proteins as well. However, while most of the immature form is associated with the complexes, very little of the mature form is. The interaction between APP and Crt is likely to be of physiological significance with respect to APP maturation since Crt is involved in quality control of nascent glycoproteins in the secretory pathway.

Increased calreticulin stability in differentiated NG-108-15 cells correlates with resistance to apoptosis induced by antisense treatment.

Since its first identification as a high-affinity calcium-binding protein over two decades ago [T.J. Ostwald and D.H. MacLennan, Isolation of a high-affinity calcium-binding protein from sarcoplasmic reticulum, J. Biol. Chem., 249 (1974) 974-979], calreticulin has become recognized as a multifunctional protein involved in a wide variety of cellular processes. We have previously shown that it has a protective function in Ca2+-mediated cell death [N. Liu, R.E. Fine, E. Simons and R.J. Johnson, Decreasing calreticulin expression lowers the Ca2+ response to bradykinin and increases sensitivity to ionomycin in NG-108-15 cells, J. Biol. Chem. , 269 (1994) 28635-28639]. We report here that in NG-108-15 neuroblastomaxglioma hybrid cells, calreticulin protein levels increase markedly when these cells are induced to differentiate by treating them with N,N-dibutyryl cAMP (db-cAMP). We demonstrate that the reason for this increase is mostly due to a large increase in the turnover time of calreticulin in differentiated cells. We also show that a calreticulin antisense oligonucleotide, CrtAS1, previously described by Liu and co-workers [N. Liu, R.E. Fine, E. Simons and R.J. Johnson, Decreasing calreticulin expression lowers the Ca2+ response to bradykinin and increases sensitivity to ionomycin in NG-108-15 cells, J. Biol. Chem., 269 (1994) 28635-28639] causes cell death in undifferentiated NG-108-15 cells when antisense treatment is extended for more than 24 h. This effect is not seen in NG-108-15 cells that have been induced to differentiate with db-cAMP until the cells have been treated with antisense for more than 4 days, due to the increased stability of Crt in these cells. Our results indicate that the mechanism by which these cells die is likely to be apoptosis.

Retinal Muller glia secrete apolipoproteins E and J which are efficiently assembled into lipoprotein particles.

We have shown that apolipoprotein E (ApoE) is synthesized by Muller cells, the major glial cell of the rabbit retina, and secreted into the vitreous after which it is taken up by retinal ganglion cells and rapidly transported into the optic nerve [Amaratunga et al., J. Biol. Chem. 271 (1996) 5628-5632]. In this report we demonstrate that the ApoE secreted by Muller cells in vivo and in culture is efficiently assembled into lipoprotein particles. Apolipoprotein J (ApoJ) is also synthesized by these cells and assembled into lipoprotein particles. The lipoproteins are triglyceride-rich and contain cholesterol esters and free cholesterol. They are heterogeneous, with densities between 1.006 and 1.18 and diameters between 14 and 45 nm. We discuss the possible role of these lipoproteins in supplying the needs of neurons for lipids, especially long axonal projection neurons such as retinal ganglion cells, which are vulnerable to age-related neurodegenerative diseases including Alzheimer's disease.