Kinetics of Rab27a-dependent actions on vesicle docking and priming in pancreatic beta-cells.
The small GTPase Rab27a, along with the isoforms of Rab3, is present on insulin secretory granules and has been implicated in regulation of Ca(2+)-triggered exocytosis. We have used membrane capacitance measurements to define the role of Rab27a in regulating the size and refilling of distinct pools of insulin granules by comparison of evoked secretory responses from Rab27a-null ashen and strain-matched wild-type control pancreatic beta-cells. We find that ashen beta-cells display a kinetic defect in refilling of readily releasable and immediately releasable vesicle pools (RRP and IRP, respectively) in response to depolarization-evoked Ca(2+) influx. The deficit in IRP refilling was not observed in the presence of stimulatory glucose concentrations (16.7 mm), though incomplete refilling of the RRP persisted. Comparatively, beta-cells from Rab3a(-/-) mice exhibited complete refilling of the IRP and RRP, demonstrating that Rab27a and Rab3a exert distinct roles in the insulin granule secretory pathway. Further, depletion of the RRP in ashen beta-cells was twofold faster than that of control beta-cells. These deficits in refilling and exocytotic rate in ashen beta-cells were absent when cAMP-regulatory pathways were activated. Elevated cAMP increased the RRP pool size, and complete refilling of the RRP occurred in ashen beta-cells; responses were comparable to wild-type controls. These effects of cAMP were largely eliminated by Rp-cAMP inhibition of PKA, indicating that PKA acts on vesicle priming downstream or via pathways independent of Rab27a. In summary, Rab27a exerts dual roles in glucose-mediated insulin granule exocytosis, facilitating refilling of releasable granule pools while also limiting the rate of release from these pools.
Merrins
Matthew J
MJ
Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109-0622, USA.
Stuenkel
Edward L
EL
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D005260:Female; D005947:Glucose / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D018345:Mice, Knockout; D015398:Signal Transduction; D020691:rab GTP-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins; D020826:rab3A GTP-Binding Protein / Q000172:deficiency / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; C486707:Rapgef4 protein, mouse; D000075745:rab27 GTP-Binding Proteins; D000242:Cyclic AMP; C512962:Rab27a protein, mouse; D020691:rab GTP-Binding Proteins; D020826:rab3A GTP-Binding Protein; D005947:Glucose
J Cell Biol. 1997 Jul 14;138(1):55-64
9214381
J Physiol. 1999 Aug 1;518 ( Pt 3):745-59
10420011
Mol Biol Cell. 2002 May;13(5):1735-49
12006666
J Cell Biol. 2005 Oct 10;171(1):99-109
16216924
J Biol Chem. 2001 Mar 30;276(13):9726-32
11134008
J Gen Physiol. 2004 Dec;124(6):641-51
15572344
J Physiol. 1999 Mar 1;515 ( Pt 2):413-7
10050008
Neuron. 2001 Apr;30(1):183-96
11343654
Cell Metab. 2006 Jun;3(6):463-8
16697276
J Gen Physiol. 2004 Dec;124(6):653-62
15572345
J Physiol. 1997 Jul 1;502 ( Pt 1):105-18
9234200
Nature. 1993 May 27;363(6427):356-8
7684514
J Cell Sci. 2006 Jun 1;119(Pt 11):2196-203
16684812
Mol Endocrinol. 1999 Feb;13(2):202-12
9973251
Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):7933-8
10859366
Mol Cell Biol. 2002 Mar;22(6):1858-67
11865063
Pflugers Arch. 2002 May;444(1-2):43-51
11976915
J Cell Biol. 2001 Feb 19;152(4):795-808
11266470
Pflugers Arch. 1992 Jan;420(1):121-3
1553260
J Physiol. 1997 Sep 1;503 ( Pt 2):399-412
9306281
FEBS Lett. 1986 Sep 15;205(2):205-9
2427366
Mol Biol Cell. 2006 Jun;17(6):2617-25
16571673
J Biol Chem. 2006 Sep 29;281(39):28657-65
16880209
Proc Natl Acad Sci U S A. 1994 May 10;91(10):4343-7
8183910
Diabetes. 1995 Dec;44(12):1461-6
7589855
Cell Signal. 2008 Jan;20(1):10-20
17716863
Neuron. 2001 Aug 30;31(4):581-91
11545717
Mol Endocrinol. 2004 Jan;18(1):117-26
14593078
Mol Cell Biol. 2002 Aug;22(15):5518-26
12101244
Diabetes. 2002 Feb;51 Suppl 1:S74-82
11815462
J Physiol. 1993 Dec;472:665-88
8145165
Diabetes. 2002 Feb;51 Suppl 1:S83-90
11815463
J Cell Biol. 2001 Feb 19;152(4):825-34
11266472
Physiol Rev. 2003 Apr;83(2):581-632
12663867
J Biol Chem. 2002 Apr 5;277(14):12432-6
11856727
J Physiol. 2008 Aug 15;586(16):3949-62
18591190
J Biol Chem. 2003 Mar 14;278(11):9715-21
12510060
J Clin Invest. 2005 Feb;115(2):388-96
15690086
J Biol Chem. 2002 Jul 12;277(28):25423-30
11980908
Science. 1996 Feb 9;271(5250):813-5
8628999
Diabetes Metab Res Rev. 2002 Nov-Dec;18(6):451-63
12469359
PLoS One. 2006 Dec 27;1:e126
17205130
Diabetologia. 1973 Apr;9(2):115-9
4577291
J Biol Chem. 2004 Mar 26;279(13):13065-75
14722103
Methods. 2004 Aug;33(4):302-11
15183179
Pflugers Arch. 2002 Jul;444(4):556-67
12136276
Neuron. 1996 Jun;16(6):1209-20
8663997
J Neurosci. 2007 Aug 8;27(32):8676-86
17687045
J Biol Chem. 1997 Nov 21;272(47):29407-10
9367993
Biophys J. 2001 Dec;81(6):3308-23
11720994
J Biochem. 2005 Jan;137(1):9-16
15713878
Proc Natl Acad Sci U S A. 2007 Dec 4;104(49):19333-8
18040047
Mol Biol Cell. 2006 May;17(5):2101-12
16481396
J Biol Chem. 2003 Mar 7;278(10):8279-85
12496249
J Biol Chem. 2003 Apr 25;278(17):15390-6
12590134
Mol Biol Cell. 2002 Jun;13(6):1906-15
12058058
Mol Biol Cell. 2003 Oct;14(10):4103-13
14517322