The Effect Of Huntingtin-associated Protein-1on Insulin Secretion And Intracellular Trafficking Of L Type Calcium Channel Cav1.2in The Pancreatic β Cells

Huntingtin-associated protein1(HAP1) was originally found to interact with Huntingtin protein (Htt), the gene product of Huntington’s disease. HAP1consists of two isoforms spliced alternately that differ at the C terminus(HAP1A and HAP1B), which is widely expressed not only in neurons, but also in the endocrine cells secreting nitrogenous hormone. HAP1participates in intracellular transport of a variety of organelles,molecular,membrane receptors and other transports in neurons. HAP1is known to interact with kinesin light chain (KLC), kinesin heavy chain (KHC), kinesin family motor protein5(KIF5), dynactin p150Glued and other microtubule-associated proteins to participate in or control the intracellular microtubule-depended transport such as the vesicle, neurotrophic factor, receptor in the nervous system. The autophosphorylation level is related to the intracellular transport of HAP1which is reflected as the following:the phosphorylation site (T598) at the C-terminal region of HaplA, which can be phosphorylated by PKA, and phosphorylation of this site can regulate the association of Hap1A with P150Glued and KLC, inhibiting its participation in the intracellular transport; while dephosphorylation shows the opposite. The molecular transport of HAP1might relate to the regulation of growth, survival and neurotransmitter release in neurons. HAP1is selectively expressed not only in the insulin secreting β cells of rat and mouse; but also in some other insulinoma cell lines such as MIN6, NIT-1, INS-1; inhibition of HAP1expression in MIN6and NIT-1cells can significantly reduce the secretion of insulin; mutant mice with Hap1deficiency in pancreatic β cells had impaired glucose tolerance and decreased insulin release in response to glucose-stimulation. All the above research indicate that HAP1is related to the insulin secretion of pancreatic β cells.Voltage gated calcium chennel(Cav) involves in the regulation of insulin secretion of pancreatic β cells stimulated by glucose. Elevation of the glucose level can promote the glucose uptake and metabolism of β cells.There after, an increase in the ATP/ADP ratio derived from glucose metabolism closes KATP channels, resulting in depolarization of the plasma membrane. The membrane depolarization in turn opens Cav channels, mediating Ca2+influx. The resultant increase in [Ca2+]i ultimately triggers the insulin secretion. Endocrine cells are mainly expressed two subtypes L-type calcium channels:Cav1.2and Cav1.3to trigger release of hormones, with Cav1.2playing an important role in β cells insulin secretion.Similar to other ion channels, newly synthesized Cav channel subunits are properly folded and assembled to build up Cav channel subunit complexes in the rough endoplasmic reticulum. Subsequently, these complexes are carried by the transport vesicles and sequentially transferred to the cis-Golgi network and the trans-Golgi network. The constitutive secretory vesicles carrying the Cav channel subunit complex separately bud off from the trans-Golgi network.Lastly,these vesicles carry Cav channel subunits to the targets, the different areas of the plasma membrane.The membrane expression level of Cav can decide the size of Ca2+influx and channel activity. Whether HAP1affects insulin secretion through its effect on intracellular trafficking and membrane expression of the calcium channels has not yet been reported.Related regulating mechanism studies of calcium channel membrane expression have shown that tumor suppressor eIF3e (eukaryotic translation initiation factor3subunit E), through its combination with Cav1.2, regulates bidirectional transport of Cavl.2in nerve cells and islet β cells, i.e. transport from cytoplasm to membrane (membrane expression) and from membrane to cytoplasm (internalization), thereby maintaining intracellular calcium homeostasis. eIF3e’s transport of Cavl.2channel and regulation of Cav1.2membrane expression indicate that HAP1might interactions with eIF3e, through which may regulate the transport of calcium channels in islet β cells, thereby affecting insulin secretion.On the basis of further confirming HAP1’s influence on β islet insulin secretion, this study observes the effect of HAP1’s expression level on Cav1.2activity, calcium influx and Cavl.2membrane expression, as well as analyzes the interactions of HAP1and eIF3e in β cells.HAP1promotes the insulin secretion of islet β cells In order to identify whether HAP1regulates the insulin secretion from pancreatic a cells, we detected the insulin levels of HAP1knockout mice and the influence of silence HAP1on the insulin secretion of rat INS-1insulinoma cells. Radioimmunoassay showed that the plasma insulin levels significantly reduced in HAP1knockout mice; Whole-cell patch-clamp membrane capacitance detection showed that, comparing the wild-type INS-1cells or the transfected HAP1-scramble plasmid INS-1cells, we found that either immediately releasable pool(IRP) secretion or rapidly releasable pool(RRP) secretion was significantly reduced in HAP1scilence INS-1cells. Phluorin is a PH-sensitive fluorescent protein, which was combinated to the N terminus of vesicle-associated membrane protein2(VAMP2), according to the PH value can trigger its changes of fluorescent during the vesicle secretion. Using VAMP2-pHluorin fluorescent probe technology and confocal live cell imaging techniques, we found that HAP1silence with HAP1-SiRNA can led to a significantly decrease of secretion frequency, secretion duration and secretion intensity, etc. of insulin vesicles.These results further confirmed the promoting effect of HAP1on pancreatic a cells insulin secretion, and the inhibition of the HAP1expression can reduce the insulin secretion of a cells.HAP1silence reduced the current and calcium influx of L-type voltage-gated calcium channel of islet a cells Calcium influx could trigger and regulate insulin secretion. In order to find out whether the influence of HAP1on insulin secretion associates with its influence on L-type calcium channels and calcium influxin in islet a cells, we tested the influence of silence HAP1on L-type calcium currents and calcium influx using INS-1cells. With replacement of calcium by barium, we used the whole-cell patch-clamp technique to record the L-type calcium current of the INS-1cells. INS-1cells were transfected with HAP1-SiRNA plasmid or HAP1-Scramble plasmid. Fourty-eight hours later, we recorded the L-type calcium current changes occurring in two experimental groups. The results demonstrated that lack of HAP1impaired L-type Ca2+current. Using Fura-2quantitative determination of intracellular calcium technology to detect the intracellular calcium concentration in INS-1cells, we found that, under conditions of high K+stimulation, the calcium concentration increased significantly in wild-type or control plasmid transfected INS-1cells, with a slight increase in the HAP1silence cells transfected with SiRNA. All the results above proved that HAP1could affect calcium influx by regulating L-type calcium channels activity of a cells, thus result in a influence on insulin secretion.HAP1silence reduced the membrane expression of Cav1.2The membrane expression level of calcium channels is one of the factors that can affect the calcium currents and concentration. With a significant decrease of the L-type calcium currents and calcium influx in INS-1cells after silencing HAP1, we suggested that HAP1might affect the membrane expression of L-type calcium channel in islet a cells. Membrane expression of ion channels is, on one hand, related to its total expression level, and on the other hand, related to its transport from the cytoplasm to the membrane. We first examined whether there was any change in mRNA expression of Cav1.2after silencing HAP1in INS-1cells. Cells transfected with HAP1-SiRNA plasmids and control plasmid, fourty-eight hours later, we examined the mRNA level of Cav1.2by using RT-PCR method and found that there was no significant difference between SiRNA cells and Control cells. In order to investigate whether HAP1affects the membrane expression through its influence on the Cav1.2transport from cytoplasm to membrane, we used the immunofluorescence techniques to analyze the HAP1’s effect on Cav1.2containing-vesicle transport and membrane expression in INS-1cells. We conexpressed YFP-Cav1.2-HA(a plasmid of Cav1.2containing a YFP fused to the intracellular N terminus,and a HA in the extracellular S5-H5loop of domain Ⅱ) and auxiliary subunits alb+a2a, together with HAP1-SiRNAor HAP1-Scramble in INS-1cells, using HA antibody immunofluorescence to investigate the membrane expression without Triton X-100, we found that silence HAP1can significantly reduced the HA immunefluorescence intensity expressed on the membrane of INS-1cells, indicating that HAP1involved in the transport of Cav1.2calcium channels from cytoplasm to membrane.The interaction between HAP1and eIF3e In order to investigate the possibility of HAP1effect on Cav1.2membrane expression in β cells through an interaction with eIF3e,which can regulate the intracellular transport of Cav1.2, we explored the interaction between HAP1and eIF3e. We used the double-labeling immunofluorescence technology to detect the localization of endogenous HAP1and eIF3e, and found that both proteins were diffusely distributed in the cytoplasm. Both eIF3e-CFP and HAP1A-YFP were coexpressed in HEK293cells and INS-1cells, and displayed an granular distribution with an significant colocation in the granulars, except a small amount of disperse distribution.Co-immunoprecipitation experiments found that there was an interaction between transfected eIF3e-Myc with HAP1A/B-GFP in HEK293, and endogenous eIF3e with HAP1in INS-1cells.This study showed that, in islet β cells, HAP1can promotes the membrane expression of L-type calcium channel, such as Cav1.2, through its interaction with eIF3e, thus increasing Cav1.2channel mediated calcium current and calcium influx and promoting the secretion of insulin.