Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells.
The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell-specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.
Yang
X C
XC
Division of Biology, California Institute of Technology, Pasadena 91125.
Labarca
C
C
Nargeot
J
J
Ho
B Y
BY
Elroy-Stein
O
O
Moss
B
B
Davidson
N
N
Lester
H A
HA
D000818:Animals; D001923:Brain Chemistry; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004330:Drosophila / Q000235:genetics; D015870:Gene Expression; D006352:Heart Ventricles / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology; D051379:Mice; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D051381:Rats; D015222:Sodium Channels / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology*; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics
D015222:Sodium Channels; D013779:Tetrodotoxin; D004247:DNA