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Rab8a is involved in membrane trafficking of Kir6.2 in the MIN6 insulinoma cell line

  • Keiichiro Uchida
  • Masatoshi Nomura
  • Tadashi Yamamoto
  • Yoshihiro Ogawa
  • Noriyoshi TeramotoEmail author
Ion channels, receptors and transporters
  • 54 Downloads
Part of the following topical collections:
  1. Ion channels, receptors and transporters

Abstract

Although ATP-sensitive K+ (KATP) channels play an important role in the secretion of insulin by pancreatic beta cells, the mechanisms that regulate the intracellular transport of KATP channel subunit proteins (i.e., Kir6.2 and sulfonylurea receptor 1 (SUR1)) to the plasma membrane remain uncharacterized. We investigated the possibility that an interaction between KATP channel subunit proteins and Rab8a protein, a member of the RAS superfamily, may be involved in the membrane trafficking of KATP channels. Co-immunoprecipitation and immunostaining experiments using co-expression systems with fluorescent protein-tagged Kir6.2 were carried out to identify the coupling of KATP channels and Rab8a proteins in the insulin-secreting cell line, MIN6. Rab8a protein co-localized with Kir6.2 protein, a channel pore subunit (in a granular pattern), and with insulin. Knockdown of the Rab8a gene with RNA interference using small interfering RNA systems caused reductions in the amount of total KATP and plasma membrane surface KATP channels without decreasing the messenger RNA transcription of the KATP channel subunits. Rab8a gene knockdown also enhanced glucose-induced insulin secretion. These results suggest that Rab8a may be involved in membrane trafficking of KATP channels and the maintenance of normal insulin secretion in the MIN6 pancreatic beta cell line.

Keywords

Channel trafficking Insulin Kir6.2 Pancreatic beta cell MIN6 Rab8a 

Abbreviations

AcGFP1

Aequorea coerulescens green fluorescent protein 1

BSA

Bovine serum albumin

cDNA

Complementary DNA

DMEM

Dulbecco’s modified Eagle’s medium

ER

Endoplasmic reticulum

FBS

Fetal bovine serum

GAP

GTPase-activating protein

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

GEF

Guanine nucleotide exchange factor

GTP

Guanosine triphosphate

HRP

Horseradish peroxidase

KATP channels

ATP-sensitive K+ channels

Kir6.x

Inwardly rectifying K+ channel 6 family

KRH

Krebs-Ringer HEPES

mRNA

Messenger RNA

PBS

Phosphate-buffered saline

PCR

Polymerase chain reaction

RPMI

Roswell Park Memorial Institute

SDS

Sodium dodecyl sulfate

SEM

Standard error of the mean

siRNA

Small interfering RNA

SUR

Sulfonylurea receptor

TBS

Tris-buffered saline

TBS-T

0.2% Tween 20 in Tris-buffered saline

Notes

Acknowledgments

We thank our colleagues for their valuable suggestions and discussion.

Funding information

This work was supported in part by the Japanese Society for the Promotion of Science (JSPS) KAKENHI (M Nomura, grant number 17K09885; N Teramoto, grant number 17H02111) and Grants-in-Aid for Research Fellowship for Young Science Foundation (T Yamamoto, grant number 18K15030).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Keiichiro Uchida
    • 1
    • 2
  • Masatoshi Nomura
    • 3
  • Tadashi Yamamoto
    • 1
  • Yoshihiro Ogawa
    • 2
    • 4
  • Noriyoshi Teramoto
    • 1
    • 5
    Email author
  1. 1.Department of Pharmacology, Faculty of MedicineSaga UniversitySagaJapan
  2. 2.Department of Medicine and Bioregulatory Science, Graduate School of Medical ScienceKyushu UniversityFukuokaJapan
  3. 3.Division of Endocrinology and Metabolism, Department of Internal Medicine, School of MedicineKurume UniversityKurumeJapan
  4. 4.Department of Molecular and Cellular Metabolism, Graduate School of Medical and Dental SciencesTokyo Medical and Dental UniversityTokyoJapan
  5. 5.Laboratory of Biomedical Engineering, Graduate School of Biomedical EngineeringTohoku UniversitySendaiJapan

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