Characterization of a partial cDNA clone detected by imidazoline receptor-selective antisera

The squid giant axon is a classic model system for understanding both excitable membranes and ion transport. To date, a Na+-driven Cl-HCO3− exchanger, sqNDCBE—related to the SLC4 superfamily and cloned from giant fiber lobe cDNA—is the only HCO3−-transporting protein cloned and characterized from a squid. The goal of our study was to clone and characterize another SLC4-like cDNA. We used degenerate PCR to obtain a partial cDNA clone (squid fiber clone 3, SF3), which we extended in both the 5′ and 3′ directions to obtain the full-length open-reading frame. The predicted amino-acid sequence of SF3 is similar to sqNDCBE, and a phylogenetic analysis of the membrane domains indicates that SF3 clusters with electroneutral Na+-coupled SLC4 transporters. However, when we measure pHi and membrane potential—or use two-electrode voltage clamping to measure currents—on Xenopus oocytes expressing SF3, the oocytes exhibit the characteristics of an electrogenic Na/HCO3− cotransporter, NBCe. That is, exposure to extracellular CO2/HCO3− not only causes a fall in pHi, followed by a robust recovery, but also causes a rapid hyperpolarization. The current-voltage relationship is also characteristic of an electrogenic NBC. The pHi recovery and current require HCO3− and Na+, and are blocked by DIDS. Furthermore, neither K+ nor Li+ can fully replace Na+ in supporting the pHi recovery. Extracellular Cl− is not necessary for the transporter to operate. Therefore, SF3 is an NBCe, representing the first NBCe characterized from an invertebrate.

Download Full-text

Isolation of a cDNA clone complementary to sequences for a 34-kilodalton protein which is a pp60v-src substrate

Molecular and Cellular Biology ◽

10.1128/mcb.4.9.1935-1938.1984 ◽

1984 ◽

Vol 4 (9) ◽

pp. 1935-1938

Author(s):

H G Tomasiewicz ◽

R Cook-Deegan ◽

D M Chikaraishi

Keyword(s):

Cdna Clone ◽

Chicken Embryo ◽

Kinase Activity ◽

Chicken Embryo Fibroblast ◽

Src Kinase ◽

Cell Protein ◽

Fibroblast Cells ◽

Chicken Cell ◽

Partial Cdna ◽

Partial Cdna Clone

We have isolated a partial cDNA clone containing sequences complementary to a mRNA encoding a 34- to 36-kilodalton normal chicken cell protein which is a substrate for pp60v-src kinase activity. Using this 34-kilodalton cDNA clone as a probe, we determined that the size of the 34-kilodalton mRNA was 1,100 nucleotides and the level of the 34-kilodalton RNA was the same in various tissues of mature chickens but was significantly higher in chicken embryo fibroblast cells.

Download Full-text

Isolation of a cDNA clone complementary to sequences for a 34-kilodalton protein which is a pp60v-src substrate.

Molecular and Cellular Biology ◽

10.1128/mcb.4.9.1935 ◽

1984 ◽

Vol 4 (9) ◽

pp. 1935-1938 ◽

Cited By ~ 1

Author(s):

H G Tomasiewicz ◽

R Cook-Deegan ◽

D M Chikaraishi

Keyword(s):

Cdna Clone ◽

Chicken Embryo ◽

Kinase Activity ◽

Chicken Embryo Fibroblast ◽

Src Kinase ◽

Cell Protein ◽

Fibroblast Cells ◽

Chicken Cell ◽

Partial Cdna ◽

Partial Cdna Clone

We have isolated a partial cDNA clone containing sequences complementary to a mRNA encoding a 34- to 36-kilodalton normal chicken cell protein which is a substrate for pp60v-src kinase activity. Using this 34-kilodalton cDNA clone as a probe, we determined that the size of the 34-kilodalton mRNA was 1,100 nucleotides and the level of the 34-kilodalton RNA was the same in various tissues of mature chickens but was significantly higher in chicken embryo fibroblast cells.

Download Full-text