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

  • Ion channels, receptors and transporters
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

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.

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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

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Acknowledgments

We thank our colleagues for their valuable suggestions and discussion.

Funding

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).

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Authors and Affiliations

  1. Department of Pharmacology, Faculty of Medicine, Saga University, Saga, 849-8501, Japan

    Keiichiro Uchida, Tadashi Yamamoto & Noriyoshi Teramoto

  2. Department of Medicine and Bioregulatory Science, Graduate School of Medical Science, Kyushu University, Fukuoka, 812-8582, Japan

    Keiichiro Uchida & Yoshihiro Ogawa

  3. Division of Endocrinology and Metabolism, Department of Internal Medicine, School of Medicine, Kurume University, Kurume, 830-0011, Japan

    Masatoshi Nomura

  4. Department of Molecular and Cellular Metabolism, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, 113-8510, Japan

    Yoshihiro Ogawa

  5. Laboratory of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan

    Noriyoshi Teramoto

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  1. Keiichiro Uchida
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Correspondence to Noriyoshi Teramoto.

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Uchida, K., Nomura, M., Yamamoto, T. et al. Rab8a is involved in membrane trafficking of Kir6.2 in the MIN6 insulinoma cell line. Pflugers Arch - Eur J Physiol 471, 877–887 (2019). https://doi.org/10.1007/s00424-018-02252-1

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