Inhibition of rat Na+(-)HCO3(-) cotransporter (NBCn1) function and expression by the alternative splice domain
Autor: | Han Soo, Yang, Deborah S, Cooper, Ira, Rajbhandari, Hae Jeong, Park, Soojung, Lee, Inyeong, Choi |
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Rok vydání: | 2009 |
Předmět: |
DNA
Complementary Patch-Clamp Techniques Reverse Transcriptase Polymerase Chain Reaction Sodium-Bicarbonate Symporters Xenopus Blotting Western Gene Expression Ion Channels Rats Electrophysiology Alternative Splicing Oocytes Animals ATP-Binding Cassette Transporters Female RNA Messenger Sodium-Potassium-Exchanging ATPase |
Zdroj: | Experimental physiology. 94(11) |
ISSN: | 1469-445X |
Popis: | The Na(+)-HCO(3)(-) cotransporter NBCn1 (SLC4A7) has multiple variants depending upon splice domains in the cytoplasmic amino- and carboxy-termini of the protein. In this study, we examined the role of the amino-terminal splice domain containing 123 amino acids (cassette II) in the regulation of NBCn1 function and expression. Polymerase chain reaction detected NBCn1 mRNAs containing cassette II in a variety of tissues. Two variants, NBCn1-B containing cassette II and NBCn1-E lacking cassette II, were expressed in Xenopus oocytes and assessed by two-electrode voltage clamp to measure the ionic current mediated by the transporters. The two variants showed similar current-voltage (I-V) relations when measured 3-4 days after RNA injection. Replacment of Cl() with gluconate did not affect the I-V relations. When exposed to solutions containing 20-50 mm Na(+), the current produced by NBCn1-B was slightly more positive than that produced by NBCn1-E. The two currents were similar at 100 mm Na(+). The slope conductances for the two variants were progressively increased at higher Na(+) levels, and the increases were parallel and superimposed. Measured at different time points after RNA injection, NBCn1-B produced lower conductance than NBCn1-E at 24-48 h. Protein expression of NBCn1-B was also low at these time points as determined by immunoblot of oocyte membrane preparation. Expressed in opossum kidney (OK) cells, NBCn1-E caused a 1.5-fold increase in ouabain-sensitive production of p-nitrophenol from p-phenyl phosphate compared with control preparations, whereas NBCn1-B had negligible effect. We conclude that the primary function of cassette II is to reduce NBCn1 protein expression. |
Databáze: | OpenAIRE |
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