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Paper Information

Title: 

EFFECT OF THE V364D MUTATION IN MEMBRANE ENDOCYTOSIS OF ROMK2 (KIR1.1B)

Type: POSTER
Author(s): HAJI HASHEMI S.,COOPER G.J.,WHITE S.J.
 
 
 
Name of Seminar: IRANIAN CONGRESS OF PHYSIOLOGY AND PHARMACOLOGY
Type of Seminar:  CONGRESS
Sponsor:  PHYSIOLOGY AND PHARMACOLOGY SOCIETY, MASHHAD UNIVERSITY OF MEDICAL SCIENCE
Date:  2007Volume 18
 
 
Abstract: 

Introduction: The renal potassium channel ROMK (Kir1.1) plays an important role in salt and water homeostasis by the kidney. ROMK is localized on the apical membrane in the cortical collecting duct and thick ascending limb of the nephron (Kohda et al. 1998; Xu et al. 1997).
Materials and Methods: Oocytes were isolated and then injected with approximately 1 ng of each cRNA as appropriate, encoding either in a final volume of 50 nl. Control oocytes were injected with 50nl of water. Mutations of the cytoplasmic termini of ROMK2 were constructed using the quikchange approach for site-directed mutagensis. ROMK-dependent currents were measured using TEVC at various time points as appropriate.
Results: The C-terminal domain of ROMK is involved in trafficking and endocytotic retrieval of the protein from the plasma membrane. Using the inhibitor Brefeldin A to stop insertion of new proteins into the cell membrane, we evaluated the stability of proteins already resident in the membrane. For -BFA oocytes neither Vm nor IK altered significantly over 48 hours. But as shown in Table 1: BFA caused a marked reduction of activity of ROMK2, but not of the mutant.

 

 

ROMK2+BFA

ROMK2V364D+BFA

T (hours)

Vm (mV)

iK (mA)

Vm (mV)

iK (mA)

0

-95.6±0.9

0.95±0.07

-96.7±0.8

1.00±0.7

48

-83.5±3.4*Æ

0.27±0.05*Æ

-96.8±0.6

1.17±0.09


 

Table 1: Vm and IK at 0 and 48 hours for EROMK2 and EROMK2V364D-expressing oocytes in the presence of BFA. P<0.001 cf. EROMK2V364D P<0.001 cf. t=0.
Conclusion: In oocytes expressing the V354D mutant, there was no decay in current at any time point during incubation with BFA at either 5
mM or 25mM. Our results show that a PDZ domain linked to the endocytosis of ROMK2 from the plasma membrane with the PDZ domain.

 
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