Hydropathy analysis predicts 11 transmembrane helices in the cardiac Na + /Ca 2+ exchanger. Using cysteine susceptibility analysis and epitope tagging, we here studied the membrane topology of the exchanger, in particular of the highly conserved internal K K-1 and K K-2 repeats. Unexpectedly, we found that the connecting loop in the K K-1 repeat forms a re-entrant membrane loop with both ends facing the extracellular side and one residue (Asn-125) being accessible from the inside and that the region containing the K K-2 repeat is mostly accessible from the cytoplasm. Together with other data, we propose that the exchanger may consist of nine transmembrane helices.z 1999 Federation of European Biochemical Societies.

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We examined the membrane topology and functional importance of residues in regions of the Na ؉ /Ca 2؉ exchanger NCX1 encompassing the conserved internal ␣ repeats by substituted cysteine scanning analysis and kinetic analysis of site-directed mutants. The results suggest that both the ␣-1 repeat and a region encompassing the ␣-2 repeat and its immediately C-terminal segment contain reentrant loop domains, each oriented in an opposite direction with respect to the membrane. We found that single or multiple mutations of six residues including Asn-125 and conserved aspartates Asp-130, Asp-825, and Asp-829 in the ␣ repeat reentrant domains reduce the apparent affinity of the exchanger for extracellular Ca 2؉ by up to 6-fold. In contrast, the triple cysteine mutation D130C/D825C/D829C did not influence the current-voltage (I-V) relationship of the exchange current. Cysteine accessibility scanning with different thiol modifiers suggested that N125C, D130C, and D825C may be located in a restricted aqueous space in the membrane accessible only to ions when examined with external probes, although N125C and D825C were previously shown to be internally accessible during exchange reaction. The results suggest that these reentrant domains in the ␣ repeats may participate in the formation of the ion transport pathway in the exchanger with some of the aspartates possibly lining it or located close to it.

In the Streptozotocin-induced diabetic rat heart, a decrease in the conductivity and suppression of electrical cell-to-cell coupling were observed. To clarify this mechanism, the present study was performed to investigate alterations of the gap junction connexin 43 (Cx43) using immunoblotting, immunohistochemistry, electron-microscopic analyses. An enhanced activation of PKCepsilon, an augmentation of PKCepsilon-mediated phosphorylation of Cx43, a decrease in the total amount of Cx43, a reduction in the area of immunoreactive particles for Cx43 at the intercalated disk, distribution of Cx43 to cell periphery or cytoplasm and the internalization approximately annular profiles of the gap junction were all characteristically recognized in the diabetic heart. Such abnormalities in the expression of Cx43 were alleviated by treatment with either lysosomal (NH(4)Cl, Leupeptin) or proteasomal inhibitor (ALLN). These results suggest that the PKCepsilon-mediated hyperphosphorylation of Cx43 makes Cx43 vulnerable to proteolytic degradation and that a decrease in the conductivity in the diabetic heart is also caused by a decrease in the number of gap junction channels due to an acceleration of the proteolytic degradation of Cx43. The remodeling of Cx43 induced by the activation of PKCepsilon may therefore contribute to the formation of the arrhythmogenic substrate in the diabetic heart. The cardioprotective effect of the remodeling of Cx43 by PKCepsilon is discussed.

1. The perforated patch method and amperometry were used to determine whether the adrenal medullary cell itself is capable of sensing hypoxia and, if so, how such sensation is transduced to secretion of catecholamines (CA). 2. Exposure to hypoxia, cyanide (CN), or muscarine facilitated CA secretion from dissociated chromaffin cells. The CN-induced secretion was not affected by removal of glucose, indicating that the CN release is due to chemical hypoxia. 3. The secretions induced by CN and muscarine were markedly diminished by removal of Ca¥ ions or by application of Cd¥ or methoxyverapamil (D_600). 4. Cyanide and muscarine produced depolarizations with generation of action potentials and increased intracellular Ca¥ concentrations determined using the acetoxymethyl (AM) ester form of fluo-3 in the presence of external Ca¥ ions, but not in their absence. 5. Hypoxia and CN produced inward currents at an equilibrium potential for Cl¦ ions, irrespective of whether or not Na¤ ions were present in the cells, and substitution of N_methyl-ª_glucamine for 134 mÒ Na¤ ions in the perfusate inhibited the CN current by 71%. The reversal potential for the CN current was −24 mV in the standard perfusate. 6. The hypoxia-, CN-and muscarine-induced currents decreased in parallel with hyperpolarizations, and exposure to CN prevented muscarine, but not nicotine, from inducing a further inward current. 7. We conclude that hypoxia and CN induce CA secretion through depolarization and the subsequent activation of voltage-dependent Ca¥ channels and that this depolarization is due to opening of cation channels, which are possibly identical to muscarinic cation channels.