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Na,K-ATPase is an ion transporter that impacts neural and glial physiology by direct electrogenic activity and the modulation of ion gradients. Its three isoforms in brain have cell-type and development-specific expression patterns. Interestingly, our studies demonstrate that in late gestation, the alpha2 isoform is widely expressed in neurons, unlike in the adult brain, in which alpha2 has been shown to be expressed primarily in astrocytes. This unexpected distribution of alpha2 isoform expression in neurons is interesting in light of our examination of mice lacking the alpha2 isoform which fail to survive after birth. These animals showed no movement; however, defects in gross brain development, muscle contractility, neuromuscular transmission, and lung development were ruled out. Akinesia suggests a primary neuronal defect and electrophysiological recordings in the pre-Bötzinger complex, the brainstem breathing center, showed reduction of respiratory rhythm activity, with less regular and smaller population bursts. These data demonstrate that the Na,K-ATPase alpha2 isoform could be important in the modulation of neuronal activity in the neonate.

The Na,K-ATPase, a member of the P-type ATPases, is composed of two subunits, alpha and beta, and is responsible for translocating Na(+) out of the cell and K(+) into the cell using the energy of hydrolysis of one molecule of ATP. The electrochemical gradient it generates is necessary for many cellular functions, including establishment of the plasma membrane potential and transport of sugars and ions in and out of the cell. Families of isoforms for both the alpha and beta subunits have been identified, and specific functional roles for individual isoforms are just beginning to emerge. The alpha4 isoform is the most recently identified Na, K-ATPase alpha isoform, and its expression has been found only in testis. Here we show that expression of the alpha4 isoform in testis is localized to spermatozoa and that inhibition of this isoform alone eliminates sperm motility. These data describe for the first time a biological function for the alpha4 isoform of the Na,K-ATPase, revealing a critical role for this isoform in sperm motility.

Using tritium suicide, we have isolated a variant of the Chinese hamster ovary cell line, CHO-K1, that is deficient in long-chain fatty alcohol:NAD+ oxidoreductase (FAO; EC 1.1.1.192). Specifically, it was the fatty aldehyde dehydrogenase component that was affected. The enzymatic deficiency found in this mutant strain, designated FAA. K1A, was similar to that displayed by fibroblasts from patients with Sjögren-Larsson syndrome (SLS), an inheritable neurocutaneous disorder. Complementation analyses suggested that the deficiency in fatty alcohol oxidation in the FAA.K1A cells and the SLS fibroblasts is a result of lesions in homologous genes. The FAA.K1A cells were unable to convert long chain fatty aldehydes to the corresponding fatty acids. This resulted in a hypersensitivity of the FAA.K1A cells to the cytotoxic effects of long chain fatty aldehydes. The difference between the mutant and wild-type cells was most obvious when using fatty aldehydes between 14 and 20 carbons, with the greatest difference between wild-type and mutant cells found when using octadecanal. Fibroblasts from a patient with SLS also displayed the hypersensitivity phenotype when compared with FAldDH+ human fibroblasts. In both CHO and human FAldDH- cell lines, addition of long chain fatty aldehydes to the medium caused a dramatic increase in aldehyde-modified phosphatidylethanolamine, presumably through Schiff's base addition to the primary amine of the ethanolamine head group. When 25 microM hexadecanal was added to the growth medium, approximately 10% of the phosphatidylethanolamine was found in the fatty aldehyde-modified form in FAA.K1A, although this was not observed in wild-type cells. Modified phosphatidylethanolamine could be detected in FAldDH- cells even when exogenous fatty aldehydes were not added to the medium. We propose a possible role for fatty aldehydes, or other aldehydic species, in mediating some of the symptoms associated with Sjögren-Larsson syndrome.

In the accompanying paper (James, P. F., and Zoeller, R. A. (1997) J. Biol. Chem. 272, 23532-23539), we reported the isolation of a series of mutants from the fibroblast-like cell line, CHO-K1, that are deficient in the incorporation of the long chain fatty alcohol, hexadecanol, into complex lipids. All but one of these mutants, FAA. K1B, were deficient in long-chain-fatty alcohol oxidase (FAO) activity. We have further characterized this FAO+ isolate. FAA.K1B cells displayed a 40% decrease in [9,10-3H]hexadecanol uptake when compared with the parent strain. Although incorporation of hexadecanol into the phospholipid fraction was decreased by 52%, the cells accumulated label in alkylglycerol (20-fold over wild type). The increase in 1-alkylglycerol labeling corresponded to a 4-fold increase in alkylglycerol mass. Short term labeling with 32Pi showed a 45-50% decrease in overall phospholipid biosynthesis in FAA.K1B. Both diacyl- and ether-linked species were affected, suggesting a general defect in phospholipid biosynthesis. Mutant cells were able to partially compensate for the decreased biosynthesis by decreasing the turnover of the phospholipid pools. The primary lesion in FAA. K1B was identified as a 95% reduction in acyl/alkyl-dihydroxyacetone-phosphate reductase activity. Whole cell homogenates from FAA.K1B were unable to reduce either acyl-dihydroxyacetone phosphate (DHAP) or alkyl-DHAP, supporting the notion that the reduction of these two compounds is catalyzed by a single enzyme. These data suggest that the biosynthesis of diacyl phospholipids, in Chinese hamster ovary cells, begins with the acylation of dihydroxyacetone phosphate as well as glycero-3-phosphate and that the "DHAP pathway" contributes significantly to diacyl glycerolipid biosynthesis. Also, the severe reduction in acyl/alkyl-DHAP reductase activity in FAA.K1B resulted in only a moderate decrease in ether lipid biosynthesis. These latter data together with the observed increase in alkylglycerol levels support the existence of a shunt pathway that is able to partially bypass the enzymatic lesion.