A carboxy-terminal valine influences endocytosis of the ROMK2 potassium channel in Xenopus laevis oocytes

  • Home
  • A carboxy-terminal valine influences endocytosis of the ROMK2 potassium channel in Xenopus laevis oocytes

University of Manchester (2003) J Physiol 552P, P108

Communications: A carboxy-terminal valine influences endocytosis of the ROMK2 potassium channel in Xenopus laevis oocytes

S. Hajihashemi*, G.J. Cooper* and S.J. White†

* Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK and † School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK

View other abstracts by:

Several studies have shown that C-terminal internalization motifs within ROMK (Kir1.1) channels are involved in clathrin-dependent endocytosis of ROMK from the plasma membrane of Xenopus laevis oocytes and rat cortical collecting duct (Moral et al. 2001; Zeng et al. 2002). We tested the involvement of valine 364, which lies within a type I PDZ-domain (EVDETDDQM) of rat ROMK2 by comparing endocytotic removal of wild-type EGFP-tagged ROMK2 (EROMK2) with a mutant (EROMK2V364D) in which V364 was replaced by aspartate via site-directed mutagenesis.

Stage V and VI oocytes isolated from humanely killed Xenopus laevis (n = 3) were injected with 50 nl H2O containing 1 ng (0.02 ng nl-1) of cRNA encoding EROMK2 or EROMK2V364D and incubated at 18 °C for 3 days. Then half the oocytes in each group were exposed continuously to 25 µM brefeldin-A (BFA), an inhibitor of protein trafficking to the plasma membrane. Following addition of BFA, function of EROMK2 and EROMK2V364D (in -BFA (n = 13-15) and +BFA (n = 13-20) oocytes) was determined in the presence or absence of 5 mM BaCl2 at an external [K+] of 2.00 mM by two-microelectrode voltage clamp. Membrane potential (Vm) and Ba2+-sensitive current (IK) are given as means ± S.E.M. and statistical significance (by ANOVA and Tukey’s test) was taken at P < 0.05.

For -BFA oocytes neither Vm nor IK altered significantly over 48 h. But as shown in Table 1, BFA caused a marked reduction of activity of EROMK2, but not of the mutant.

We conclude that wild-type EROMK2 is endocytosed from the plasma membrane of Xenopus oocytes and that substitution of valine 364 by aspartate renders the protein resistant to endocytosis. These results are consistent with the idea that interaction between the C-terminus of ROMK2 and components of the endocytotic mechanism allows entry of ROMK2 into the endocytotic pathway and such interaction may require a functional PDZ domain.

SH is a graduate student supported by the Iranian Ministry of Health and Medical Education and the Arak University of Medical Sciences.

Table 1. Vm and IK at 0 and 48 h for EROMK2- and EROMK2V364D-expressing oocytes in the presence of BFA.Symbols indicate *P < 0.001 cf. EROMK2V364D, †P < 0.001cf. t = 0
Click to enlarge

Table 1. Vm and IK at 0 and 48 h for EROMK2- and EROMK2V364D-expressing oocytes in the presence of BFA.Symbols indicate *P &lt; 0.001 cf. EROMK2V364D, †P &lt; 0.001cf. t = 0

Where applicable, experiments conform with Society ethical requirements.

Menu Menu Search

Site search

Filter

Content Type