Structure/Function Analysis of human ZnT8 (SLC30A8): A Diabetes Risk Factor and Zinc Transporter

Abstract

The human zinc transporter ZnT8 (SLC30A8) is expressed primarily in pancreatic β-cells and plays a key function in maintaining the concentration of blood glucose through its role in insulin storage, maturation and secretion. ZnT8 is an autoantigen for Type 1 diabetes (T1D) and is associated with Type 2 diabetes (T2D) through its risk allele that encodes a major non-synonymous single nucleotide polymorphism (SNP) at Arg325. Loss of function mutations improve insulin secretion and are protective against diabetes. Despite its role in diabetes and concomitant potential as a drug target, little is known about the structure or mechanism of ZnT8. To this end, we expressed ZnT8 in Pichia pastoris yeast and Sf 9 insect cells. Guided by a rational screen of 96 detergents, we developed a method to solubilize and purify recombinant ZnT8. An in vivo transport assay in Pichia and a liposome-based uptake assay for insect-cell derived ZnT8 showed that the protein is functionally active in both systems. No significant difference in activity was observed between full-length ZnT8 (ZnT8A) and the amino-terminally truncated ZnT8B isoform. A fluorescence-based in vitro transport assay using proteoliposomes indicated that human ZnT8 functions as a Zn 2+ /H + antiporter. We also purified E. coli -expressed amino- and carboxy-terminal cytoplasmic domains of ZnT8A. Circular dichroism spectrometry suggested that the amino-terminal domain contains predominantly α-helical structure, and indicated that the carboxy-terminal domain has a mixed α/β structure. Negative-stain electron microscopy and single-particle image analysis yielded a density map of ZnT8B at 20 Å resolution, which revealed that ZnT8 forms a dimer in detergent micelles. Two prominent lobes are ascribed to the transmembrane domains, and the molecular envelope recapitulates that of the bacterial zinc transporter YiiP. These results provide a foundation for higher resolution structural studies and screening experiments to identify compounds that modulate ZnT8 activity.

Probing the Structure and Function of the Cytosolic Domain of the Human Zinc Transporter ZnT8 with Nickel(II) Ions

“…The CTD forms a tetramer when the N-terminal 6xHis tag is removed [ 39 ]. In agreement with the investigations of others, we find that the CTD with the tag forms a dimer [ 19 , 21 ]. Investigating Ni(II) binding is also important for the biology of this transporter, because there are Ni 2+ transporters in the CDF family [ 40 ] and nickel has been found in human islets [ 41 ], and therefore selectivities of the metal binding sites need to be understood.…”

“…Specifically, for ZnT8, we are also investigating how the W/R mutation at the apex of the CTD of ZnT8 affects metal transport far away in the transmembrane domain and thus can modulate the risk to developing type 2 diabetes. Two recent cryo-EM structures of full-length human ZnT8 describe its overall structure [ 19 , 20 ]. One of the two investigations provides a model at relatively high resolution (3–5 Å) and important new information on metal binding [ 20 ].…”

“…Two cryo-EM investigations of human ZnT8, one at relatively low resolution (about 20 Å) and another at relatively high resolution (about 4 Å), have now provided additional insights [19,20] and confirm the differences we noted in zinc binding of the CTD compared to the bacterial YiiP protein and the involvement of the C-terminal cysteines in zinc binding [21]. The metal sensing mechanism of the CTD discussed for the YiiP protein is based on the presence of two pairs of binuclear metal ion sites at the dimer interface.…”

Inside the Insulin Secretory Granule

“…Secretory pathway Zn 2+ uptake is under the control of the Zn 2+ transporter (ZnT) family. ZnTs dimerize to localize and function and likely do so as H + /Zn 2+ antiporters [ 246 , 257 ], so therefore the establishment of a luminal proton gradient by V-ATPase may be permissive for Zn 2+ uptake. ZnT5 and Znt7 localize to the β-cell Golgi apparatus whereas ZnT5 and ZnT8 are in the SG [ 28 ] ( Figure 6 ).…”

“…Finally, humans heterozygous for a ZnT8 variant truncated at residue 138, which impairs transporter synthesis and thus results in ZnT8 haploinsufficiency, are equipped with increased proinsulin conversion and circulating insulin, and improved glucose tolerance [ 270 , 271 ]. Since ZnTs are likely to function as H + /Zn 2+ exchangers [ 246 , 257 ], enhanced flux through ZnT8 could buffer the reduction in luminal pH during SG maturation to limit endoprotease activity. Accelerated packing of the granule matrix in the presence of high Zn 2+ could also reduce the accessibility of proinsulin to the endoproteases for conversion.…”