Functional expression of receptors for vasoactive intestinal contractor (VIC) in Xenopus oocytes.

Native Xenopus oocytes express two isoforms of muscarinic receptors that mediate qualitatively different physiological responses. Oocytes of the majority of donors (common) express M3-like receptors (M3Rs) at comparable densities at both the animal and vegetal hemispheres of the cell. Rare (variant) donors possess oocytes that express mainly M1-like receptors (M1Rs), localized predominantly at the animal hemisphere. We have investigated the apparent degradation of these two isoforms and its relationship to their hemispheric distribution. Cycloheximide (CHX) caused a time-dependent decrease in receptor-mediated responses and [3H]quinuclidinyl benzylate (QNB) binding in oocytes from both types of donors. The t1/2 values ranged between 3 and 7 h. Removal of CHX resulted in rapid recovery of the response. This implied rapid degradation and turnover of both types of receptors. The loss of M1Rs was more than that of M3Rs. Moreover, the decrease was more rapid and more extensive on the animal hemisphere in both types of donors. Injection of oocytes expressing either receptor isoform with specific antisense oligonucleotides complementary to either m1 or m3 muscarinic receptors (from mouse) showed receptor loss at approximately the same rate as that calculated from experiments with CHX. Furthermore, oocytes of variant donors express M1Rs exclusively on the animal hemisphere, while the residual activity found on the vegetal hemisphere of the cell was mediated by M3Rs. Inhibition of putative receptor glycosylation with tunicamycin caused a rapid decrease in receptor-mediated responses and radioligand binding on M1Rs, but had virtually no effect on M3Rs. The expression of cloned m1 muscarinic receptors, however, was not affected by tunicamycin, suggesting that glycosylation is not a general prerequisite for the functional expression of muscarinic receptors.

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Functional expression of a vertebrate inwardly rectifying K+ channel in yeast.

Molecular Biology of the Cell ◽

10.1091/mbc.6.9.1231 ◽

1995 ◽

Vol 6 (9) ◽

pp. 1231-1240 ◽

Cited By ~ 51

Author(s):

W Tang ◽

A Ruknudin ◽

W P Yang ◽

S Y Shaw ◽

A Knickerbocker ◽

...

Keyword(s):

Transcriptional Control ◽

Xenopus Oocytes ◽

Genetic System ◽

Functional Expression ◽

Inward Rectifier ◽

K Channel ◽

Coding Region ◽

Low Potassium ◽

K Current ◽

Inwardly Rectifying

We describe the expression of gpIRK1, an inwardly rectifying K+ channel obtained from guinea pig cardiac cDNA. gpIRK1 is a homologue of the mouse IRK1 channel identified in macrophage cells. Expression of gpIRK1 in Xenopus oocytes produces inwardly rectifying K+ current, similar to the cardiac inward rectifier current IK1. This current is blocked by external Ba2+ and Cs+. Plasmids containing the gpIRK1 coding region under the transcriptional control of constitutive (PGK) or inducible (GAL) promoters were constructed for expression in Saccharomyces cerevisiae. Several observations suggest that gpIRK1 forms functional ion channels when expressed in yeast. gpIRK1 complements a trk1 delta trk2 delta strain, which is defective in potassium uptake. Expression of gpIRK1 in this mutant restores growth on low potassium media. Growth dependent on gpIRK1 is inhibited by external Cs+. The strain expressing gpIRK1 provides a versatile genetic system for studying the assembly and composition of inwardly rectifying K+ channels.

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Cloning of a human B2 bradykinin receptor: Functional expression and pharmacological characterization in Xenopus oocytes and mammalian cell lines

Peptides ◽

10.1007/978-94-011-0683-2_143 ◽

1994 ◽

pp. 433-434

Author(s):

E. A. Novotny ◽

M. A. Connolly ◽

S. Chakravarty ◽

S. Lu ◽

D. E. Wilkins ◽

...

Keyword(s):

Cell Lines ◽

Mammalian Cell ◽

Xenopus Oocytes ◽

Functional Expression ◽

Pharmacological Characterization ◽

Mammalian Cell Lines ◽

Bradykinin Receptor

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Functional expression of nerve terminal in Xenopus oocytes.

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)50130-5 ◽

1994 ◽

Vol 64 ◽

pp. 122

Author(s):

Hirotomo Shibaguchi ◽

Yasufumi Kataoka ◽

Keiko Takemura ◽

Shigeru Yoshida ◽

Masami Kouzuma ◽

...

Keyword(s):

Nerve Terminal ◽

Xenopus Oocytes ◽

Functional Expression

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Roles of Slc13a1 and Slc26a1 sulfate transporters of eel kidney in sulfate homeostasis and osmoregulation in freshwater

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00725.2004 ◽

2005 ◽

Vol 289 (2) ◽

pp. R575-R585 ◽

Cited By ~ 25

Author(s):

Tsutomu Nakada ◽

Kambiz Zandi-Nejad ◽

Yukihiro Kurita ◽

Hisayuki Kudo ◽

Vadjista Broumand ◽

...

Keyword(s):

Xenopus Oocytes ◽

Northern Blot Analysis ◽

Functional Expression ◽

Sulfate Concentration ◽

Sulfate Transport ◽

Dual Roles ◽

Regulated Expression ◽

Freshwater Eel ◽

Sat 1

Sulfate is required for proper cell growth and development of all organisms. We have shown that the renal sulfate transport system has dual roles in euryhaline eel, namely, maintenance of sulfate homeostasis and osmoregulation of body fluids. To clarify the physiological roles of sulfate transporters in teleost fish, we cloned orthologs of the mammalian renal sulfate transporters Slc13a1 (NaSi-1) and Slc26a1 (Sat-1) from eel ( Anguilla japonica) and assessed their functional characteristics, tissue localization, and regulated expression. Full-length cDNAs coding for ajSlc13a1 and ajSlc26a1 were isolated from a freshwater eel kidney cDNA library. Functional expression in Xenopus oocytes revealed the expected sulfate transport characteristics; furthermore, both transporters were inhibited by mercuric chloride. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated robust apical and basolateral expression of ajSlc13a1 and ajSlc26a1, respectively, within the proximal tubule of freshwater eel kidney. Expression was dramatically reduced after the transfer of eels from freshwater to seawater; the circulating sulfate concentration in eels was in turn markedly elevated in freshwater compared with seawater conditions (19 mM vs. 1 mM). The reabsorption of sulfate via the apical ajSlc13a1 and basolateral ajSlc26a1 transporters may thus contribute to freshwater osmoregulation in euryhaline eels, via the regulation of circulating sulfate concentration.

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