RalA GTPase tethers insulin granules to L- and R-type calcium channels through binding α2 δ-1 subunit.
RalA GTPase has been implicated in the regulated delivery of exocytotic vesicles to the plasma membrane (PM) in mammalian cells. We had reported that RalA regulates biphasic insulin secretion, which we have now determined to be contributed by RalA direct interaction with voltage-gated calcium (Cav ) channels. RalA knockdown (KD) in INS-1 cells and primary rat β-cells resulted in a reduction in Ca(2+) currents arising specifically from L-(Cav 1.2 and Cav 1.3) and R-type (Cav 2.3) Ca(2+) channels. Restoration of RalA expression in RalA KD cells rescued these defects in Ca(2+) currents. RalA co-immunoprecipitated with the Cav α2 δ-1 auxiliary subunit known to bind the three Cav s. Moreover, the functional molecular interactions between Cav α2 δ-1 and RalA on the PM shown by total internal reflection fluorescent microscopy/FRET analysis could be induced by glucose stimulation. KD of RalA inhibited trafficking of α2 δ-1 to insulin granules without affecting the localization of the other Cav subunits. Furthermore, we confirmed that RalA and α2 δ-1 functionally interact since RalA KD-induced inhibition of Cav currents could not be recovered by RalA when α2 δ-1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds α2 δ-1 on insulin granules to tether these granules to PM Ca(2+) channels. This acts as a chaperoning step prior to and in preparation for sequential assembly of exocyst and excitosome complexes that mediate biphasic insulin secretion.
Department of Physiology and Medicine, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
Li
L
Xie
Youhou
Y
Kang
Tao
T
Liang
Subhankar
S
Dolai
Huanli
H
Xie
Leon
L
Parsaud
Jamie A
JA
Lopez
Yu
Y
He
Subbulakshmi
S
Chidambaram
Patrick P
PP
Lam
David E
DE
James
Shuzo
S
Sugita
Herbert Y
HY
Gaisano
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D020908:Calcium Channels, R-Type / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005089:Exocytosis; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D034741:RNA, Small Interfering; D051381:Rats; D017207:Rats, Sprague-Dawley; D022142:Secretory Vesicles / Q000378:metabolism*; D020725:ral GTP-Binding Proteins / Q000378:metabolism*
D020746:Calcium Channels, L-Type; D020908:Calcium Channels, R-Type; D007328:Insulin; D021122:Protein Subunits; D034741:RNA, Small Interfering; C492197:Rala protein, rat; D020725:ral GTP-Binding Proteins; D002118:Calcium