Paul F. James

Identification of a Specific Role for the Na,K-ATPase α2 Isoform as a Regulator of Calcium in the Heart

It is well accepted that inhibition of the Na,K-ATPase in the heart, through effects on the Na/Ca exchanger, raises the intracellular Ca2+ concentration and strengthens cardiac contraction. However, the contribution that individual isoforms make to this calcium regulatory role is unknown. Assessing the phenotypes of mouse hearts with genetically reduced levels of Na,K-ATPase alpha 1 or alpha 2 isoforms clearly demonstrates different functional roles for these isoforms in vivo. Heterozygous alpha 2 hearts are hypercontractile as a result of increased calcium transients during the contractile cycle. In contrast, heterozygous alpha 1 hearts are hypocontractile. The different functional roles of these two isoforms are further demonstrated since inhibition of the alpha 2 isoform with ouabain increases the contractility of heterozygous alpha 1 hearts. These results definitively illustrate a specific role for the alpha 2 Na,K-ATPase isoform in Ca2+ signaling during cardiac contraction.

Sperm Motility Is Dependent on a Unique Isoform of the Na,K-ATPase

The Na,K-ATPase, a member of the P-type ATPases, is composed of two subunits, α and β, 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 α and β subunits have been identified, and specific functional roles for individual isoforms are just beginning to emerge. The α4 isoform is the most recently identified Na,K-ATPase α isoform, and its expression has been found only in testis. Here we show that expression of the α4 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 α4 isoform of the Na,K-ATPase, revealing a critical role for this isoform in sperm motility.

The Na,K-ATPase α2 Isoform Is Expressed in Neurons, and Its Absence Disrupts Neuronal Activity in Newborn Mice

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 α2 isoform is widely expressed in neurons, unlike in the adult brain, in which α2 has been shown to be expressed primarily in astrocytes. This unexpected distribution of α2 isoform expression in neurons is interesting in light of our examination of mice lacking the α2 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 α2 isoform could be important in the modulation of neuronal activity in the neonate.

Characterization of the Fourth α Isoform of the Na,K-ATPase

Abstract. The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, α and β, and tissue-specific isoforms exist for each of these, α1, α2 and α3 and β1, β2 and β3. We have proposed that an additional α isoform, α4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative α4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in α4 isoform cDNA transfected 3T3 cells. Using an α4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical KD values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na+ and K+. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis.

Genetic Profiling Reveals Global Changes in Multiple Biological Pathways in the Hearts of Na, K-ATPase Alpha 1 Isoform Haploinsufficient Mice

The Na,K-ATPase transports three sodium ions out of the cell and two potassium ions into the cell using ATP hydrolysis for energy. The ion gradient formed by the Na,K-ATPase contributes to the resting membrane potential, maintains cellular excitability and is important for glucose and amino acid uptake in the cell. The α1 catalytic isoform is expressed in virtually all cell types. We have previously examined cardiac physiology of mice lacking one copy of the α1 isoform gene of the Na,K-ATPase. The observation of reduced cardiac contractility in the α1 heterozygous mice was unexpected since mice heterozygous for the α2 isoform displayed enhanced cardiac contractility similar to what would be observed with cardiac glycoside treatment. We further examined hearts from α1 heterozygous mice to identify genomic responses to reduced Na,K-ATPase capacity. Using microarray analyses, we identified groups of genes whose expressions were perturbed in the α1 heterozygous hearts compared to wild-type. Known functional relationships of these genes suggest that multiple biological pathways are altered by α1 hemizygosity including activation of the renin-angiotensin system, changes in genes of energy metabolism and transport and elevated brain natriuretic peptide. This suggests that Na,K-ATPase α1 isoform activity may be required in numerous cellular processes.