Inhibition of rat Na+(-)HCO3(-) cotransporter (NBCn1) function and expression by the alternative splice domain.
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.
Yang
Han Soo
HS
Department of Physiology, Emory University, Atlanta, GA 30322, USA.
Cooper
Deborah S
DS
Rajbhandari
Ira
I
Park
Hae Jeong
HJ
Lee
Soojung
S
Choi
Inyeong
I
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D015153:Blotting, Western; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004594:Electrophysiology; D005260:Female; D015870:Gene Expression / Q000235:genetics / Q000502:physiology; D007473:Ion Channels / Q000502:physiology; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics* / Q000502:physiology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014981:Xenopus
D018528:ATP-Binding Cassette Transporters; D018076:DNA, Complementary; D007473:Ion Channels; D012333:RNA, Messenger; C498902:Slc4a7 protein, rat; D027982:Sodium-Bicarbonate Symporters; D000254:Sodium-Potassium-Exchanging ATPase