Elsevier

Developmental Brain Research

Volume 123, Issue 2, 28 October 2000, Pages 165-172
Developmental Brain Research

Research report
Quantification of the α3 subunit of the Na+/K+-ATPase in developing rat cerebellum

https://doi.org/10.1016/S0165-3806(00)00084-5Get rights and content

Abstract

Cerebellar Purkinje neurons of rats have been shown to exhibit a progressive increase in resting membrane potential as the animals develop postnatally. The magnitude of this increase was equivalent in magnitude to the increase in the depolarizing action of ouabain, consistent with a role for the Na+/K+-pump in the hyperpolarization. Ouabain binding sites in whole cerebellum also increased with age. The present study was undertaken to confirm that the increases in ouabain binding and the electrophysiological responses to ouabain were a consequence of increases in the sodium pump and to determine whether the changes seen at the whole organ level were reflective of changes taking place at the cellular level. Using antibodies directed against the α1, α2, and α3 subunits of the Na+/K+-ATPase, rats between 13 and 19 days of age exhibited a statistically significant increase in the relative amount of the α3 subunit at the level of the whole organ, as determined by Western and slot blot analyses, with no change in the levels of either the α1 or the α2 subunit. Using immunohistochemistry, the α3 subunit was shown to increase in both the Purkinje cell layer and the white matter during this postnatal time period, while the α1 subunit increased in the granular layer. These results support and extend previous work, which pointed to a role for the electrogenic sodium pump in the developmental increase in Purkinje cell membrane potential. Furthermore, the data provide a cellular mechanism underlying the increase in resting membrane potential, that is, by the specific modulation of the α3 subunit isoform.

Introduction

The Purkinje neurons of the developing rat cerebellum exhibit changes in membrane properties as the cells mature postnatally. Of particular developmental importance is the time period between postnatal days 10 and 20, when the regular pattern of Purkinje cell firing shifts to the typical irregular pattern of firing [12], [36] and the dendritic tree develops [5]. Midway through these time-points both excitatory [12], [21], [22], [30], [36], [37] and inhibitory innervation [1], [2], [30] to Purkinje neurons have been established. Within this same time-frame a significant increase in resting membrane potential occurs that is the result of an increase in resting electrogenic Na+,K+ pumping [24]. This hyperpolarization seen at about 15 days of age is consistent with the hyperpolarizing influence of the Na+/K+-ATPase measured by Genet and Kado [16] in animals 3–5 weeks of age. Although the activity of the sodium pump can be enhanced acutely by PKA activation [8] and increased intracellular Na+ [14], chronic changes observed in developing rat cerebellum are the result of increased amounts of the enzyme [24], as has been shown for the brain as a whole [6], [28], [29].

The Na+/K+-ATPase found in the brain is a heterodimer composed of α and β subunits [33]. The α subunit binds Na+ and K+, contains the ouabain-binding site, and exhibits the ATPase activity while the β subunit regulates the Na+ and K+ affinities of the α subunit, plays a role in the occlusion of K+, and is involved in the delivery of the α subunit to the plasma membrane [7]. Of the four α and three β isoforms [7], α1, α2, and α3 and β1 and β2 are known to be expressed in adult rat cerebellum [23], [26], [35]. The α3β1 dimer is the primary one found in the Purkinje neurons of adult rats, although there is some evidence for the presence of α2 and β2 subunits in Purkinje cell dendrites [26].

The present work was undertaken in an effort to ascertain if the increase in the Na+/K+-ATPase as evidenced by [3H]-ouabain binding at the whole organ level [24] was reflective of the changes taking place at the cellular level and could be ascribed to specific molecular modifications. Using antibodies directed against the three α subunits, we present both qualitative and quantitative evidence of an increase in the level of the α3 subunit isoform of the sodium pump in the Purkinje neurons of the developing rat cerebellum which correlates temporally to the increased membrane potential seen in these cells.

Section snippets

Animals

Thirteen- and 19-day-old Sprague–Dawley rats were sacrificed via decapitation in accordance with procedures approved by the Animal Care and Use Committee. All efforts were employed to minimize animal suffering.

Antibodies

The antibodies used against the α subunits of the Na+/K+-ATPase were monoclonals of mouse origin. The antibody against the α1 subunit was M8-P1-A3 (Affinity Bioreagents, Inc.) [4]. The antibody against the α2 subunit was McB2 (the generous gift of Dr Kathleen J. Sweadner [25]. The

Results

In order to measure changes in the level of the sodium pump, antibodies were used which were directed against the three isoforms of the α subunit. Initial studies were undertaken to determine if there were changes in the amount of the three subunit isoforms at the level of the entire cerebellum. Fig. 1 shows the results of two Western blots. Between 13 and 19 days there is a substantial increase in the amount of the α3 subunit. There appears to be no α2 subunit detectable in either 13 or 19 day

Discussion

Radioligand binding studies [24] using [3H]-ouabain pointed to a postnatal increase in total Na+-pump α subunits in the cerebellum of rats between 10 and 14 and 15+ days. This supported evidence from electrophysiological experiments [24] that, during the same period, there was a hyperpolarization of Purkinje neurons related to enhanced electrogenic activity of the Na+,K+ pump. In the present studies, Western blot and slot blot analysis using isoform specific antibodies increased the level of

Acknowledgements

Special thanks to Dr Albert Berrebi, Dr Richard Dey, Brian Satterfield, Tanya Mulvey, Jeff Altemus, and Allison Reed. Supported in part by a grant from the National Institutes of Drug Abuse (DA03773) to Dr David A. Taylor and Dr William W. Fleming and (DA03773S1) to Dr David A. Taylor and Dr Peggy S. Biser.

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