Developmental regulation of sodium channel expression in the rat forebrain

Abstract

Na+ channels in adult rat brain are heterotrimeric complexes consisting of alpha subunits (260 kDa) noncovalently associated with a beta 1 subunit (36 kDa) and disulfide-linked to a beta 2 subunit (33 kDa). The time course of developmental accumulation of the 9-kilobase mRNA encoding sodium channel alpha subunits in the rat forebrain was measured by RNA blotting. These transcripts were present at low levels until birth, increased rapidly in abundance to peak by postnatal day 7, and subsequently declined to 50% of this maximum value in adult animals. Sodium channel gene transcription measured by a nuclear run-on assay was first detectable on embryonic day 16, increased to a maximum on postnatal days 1 through 7, and declined in adulthood. The level of gene transcription was highest during the period of rapid rise of Na+ channel alpha subunit mRNA levels and decreased during the period of Na+ channel mRNA decline. The levels of Na+ channel alpha subunit protein measured by immunoblotting increased from postnatal day 1 to postnatal day 21, with the greatest rate of increase falling between days 7 and 21. The number of high affinity saxitoxin binding sites increased in parallel to the increase in alpha subunit protein. The period of most rapid rise in Na+ channel alpha subunit levels corresponded to the period of greatest Na+ channel mRNA abundance. Na+ channel alpha subunits were resolved into free alpha subunits and alpha subunits disulfide-linked to beta 2 subunits. On postnatal day 1, virtually all Na+ channel alpha subunits were in the free alpha form. The fraction of disulfide-linked alpha subunits increased to 60% by postnatal day 21 and 90% by postnatal day 90. The concentration of free alpha subunits was maximum on postnatal days 7 to 14 and declined to less than 10% in adulthood. We conclude from these data that the formation of mature heterotrimeric sodium channel complexes is regulated by at least two processes in developing rat forebrain. Activation of Na+ channel alpha subunit gene transcription and the subsequent increase in Na+ channel mRNA are responsible for the major increases in alpha subunit protein and functional Na+ channels in the neonatal brain. However, changes in alpha subunit mRNA abundance alone are not sufficient to explain the kinetics of alpha subunit protein accumulation. Kinetic analysis suggests a requirement for a developmentally regulated translational or post-translational step in brain sodium channel expression.