Chromogranin B (CHGB) is dimorphic and responsible for dominant anion channels delivered to cell surface via regulated secretion

Gaya P. Yadav; Haiyuan Wang; Joke Ouwendijk; Stephen Cross; Qiaochu Wang; Feng Qin; Paul Verkade; Michael X. Zhu; Qiu Xing Jiang

doi:10.3389/fnmol.2023.1205516

Chromogranin B (CHGB) is dimorphic and responsible for dominant anion channels delivered to cell surface via regulated secretion

Gaya P. Yadav, Haiyuan Wang, Joke Ouwendijk, Stephen Cross, Qiaochu Wang, Feng Qin, Paul Verkade, Michael X. Zhu^*, Qiu Xing Jiang^*

^*Corresponding author for this work

Research output: Contribution to journal › Article (Academic Journal) › peer-review

2 Citations (Scopus)

Abstract

Regulated secretion is conserved in all eukaryotes. In vertebrates granin family proteins function in all key steps of regulated secretion. Phase separation and amyloid-based storage of proteins and small molecules in secretory granules require ion homeostasis to maintain their steady states, and thus need ion conductances in granule membranes. But granular ion channels are still elusive. Here we show that granule exocytosis in neuroendocrine cells delivers to cell surface dominant anion channels, to which chromogranin B (CHGB) is critical. Biochemical fractionation shows that native CHGB distributes nearly equally in soluble and membrane-bound forms, and both reconstitute highly selective anion channels in membrane. Confocal imaging resolves granular membrane components including proton pumps and CHGB in puncta on the cell surface after stimulated exocytosis. High pressure freezing immuno-EM reveals a major fraction of CHGB at granule membranes in rat pancreatic β-cells. A cryo-EM structure of bCHGB dimer of a nominal 3.5 Å resolution delineates a central pore with end openings, physically sufficient for membrane-spanning and large single channel conductance. Together our data support that CHGB-containing (CHGB+) channels are characteristic of regulated secretion, and function in granule ion homeostasis near the plasma membrane or possibly in other intracellular processes.

Original language	English
Article number	1205516
Journal	Frontiers in Molecular Neuroscience
Volume	16
DOIs	https://doi.org/10.3389/fnmol.2023.1205516
Publication status	Published - 26 Jun 2023

Bibliographical note

Funding Information:
We are grateful to Dr. Barbara Ehrlich (Yale University) for sharing a construct for mouse CHGB, to Dr. Herbert Y. Gaisano (University of Toronto) for a syncollin-pHluorin construct, to Dr. Kuixing Zhang (UC San Diego) for sharing his techniques for releasing granular contents from endocrine cells, to Dr. Wen-Hong Li at UT Southwestern Medical Center for sharing the murine INS-1 cells and the insulin detection methods, and to Dr. Jerry Shay for providing the Lentiviral construct of CHGB shRNA. We thank Dr. Mahesh Chandak and Ms. Sutonuka Bhar (then at the University of Florida) for their technical assistance, and Prof. Tsan Sam Xiao at Case Western Reserve University for his advice and assistance on modeling from the cryo-EM maps. Special thanks go to Drs. Thomas Klose and Wen Jiang at the Purdue University, Drs. Ken Taylor, Xiaofeng Zheng, Nilakshee Battacharya and others at Florida State University, Drs. Ulrich Baxa, Thomas Edwards, Adam Wier and others at the National Cryo-EM Facility (NCEF) of National Cancer Institute (NCI) for their technical support in data collection in their microscopes. Isolated rat islets for high pressure freezing experiments were kindly provided by Prof. Michele Solimena at Technical University Dresden, Germany (to the Verkade lab). Some of the bilayer experiments were performed in a rig assembled in Dr. Feng Qin’s laboratory in the University at Buffalo. We thank Drs. Jerry Shay (UT Southwestern), Ken Taylor (Florida State University), Wen Jiang (Purdue University) and Sixue Chen (University of Florida) for working with Q-XJ on joint grants related to this work.

Funding Information:
The work in the Jiang lab was supported by NIH (R21GM131231, R01GM111367, and R01GM093271 to Q-XJ), CF Foundation (JIANG15G0 to Q-XJ), Welch Foundation (I-1684 to Q-XJ) and CPRIT (RP120474 to Q-XJ), and by an AHA National Innovative Award (12IRG9400019 to Q-XJ), an NIGMS EUREKA Award (R01GM088745 to Q-XJ), a pilot grant from the Office of Research at the University of Florida (to Q-XJ), and startup funds from the University of Texas Southwestern Medical Center, the University of Florida and the Hauptman-Woodward Medical Research Institute. Some of the experiments reported here were performed in a laboratory constructed with support from NIH (C06RR30414). Besides EM facilities at home institutions, the cryo-EM studies in the Jiang lab were supported by SEM4 consortium at the Florida State University (grant #1U24GM116788) with Q-XJ as one of the MPIs, the National Cancer Institute’s National Cryo-EM Facility (NCEF) at the Frederick National Laboratory for Cancer Research under a contract HSSN261200800001E, the NIH-funded consortium at the Purdue University (U24GM116789) with Q-XJ as one of the co-PIs as well as facilities at the Case Western Reserve University and the New York Structural Biology Center under the support of the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24GM129539) and by grants from the Simons Foundation (SF349247) and NY State Assembly Majority.

Publisher Copyright:
Copyright © 2023 Yadav, Wang, Ouwendijk, Cross, Wang, Qin, Verkade, Zhu and Jiang.

Keywords

cell surface anion channels
cryo-EM structures
dimorphic CHGB
regulated secretory pathways
secretory granule exocytosis

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title = "Chromogranin B (CHGB) is dimorphic and responsible for dominant anion channels delivered to cell surface via regulated secretion",

abstract = "Regulated secretion is conserved in all eukaryotes. In vertebrates granin family proteins function in all key steps of regulated secretion. Phase separation and amyloid-based storage of proteins and small molecules in secretory granules require ion homeostasis to maintain their steady states, and thus need ion conductances in granule membranes. But granular ion channels are still elusive. Here we show that granule exocytosis in neuroendocrine cells delivers to cell surface dominant anion channels, to which chromogranin B (CHGB) is critical. Biochemical fractionation shows that native CHGB distributes nearly equally in soluble and membrane-bound forms, and both reconstitute highly selective anion channels in membrane. Confocal imaging resolves granular membrane components including proton pumps and CHGB in puncta on the cell surface after stimulated exocytosis. High pressure freezing immuno-EM reveals a major fraction of CHGB at granule membranes in rat pancreatic β-cells. A cryo-EM structure of bCHGB dimer of a nominal 3.5 {\AA} resolution delineates a central pore with end openings, physically sufficient for membrane-spanning and large single channel conductance. Together our data support that CHGB-containing (CHGB+) channels are characteristic of regulated secretion, and function in granule ion homeostasis near the plasma membrane or possibly in other intracellular processes.",

keywords = "cell surface anion channels, cryo-EM structures, dimorphic CHGB, regulated secretory pathways, secretory granule exocytosis",

author = "Yadav, {Gaya P.} and Haiyuan Wang and Joke Ouwendijk and Stephen Cross and Qiaochu Wang and Feng Qin and Paul Verkade and Zhu, {Michael X.} and Jiang, {Qiu Xing}",

note = "Funding Information: We are grateful to Dr. Barbara Ehrlich (Yale University) for sharing a construct for mouse CHGB, to Dr. Herbert Y. Gaisano (University of Toronto) for a syncollin-pHluorin construct, to Dr. Kuixing Zhang (UC San Diego) for sharing his techniques for releasing granular contents from endocrine cells, to Dr. Wen-Hong Li at UT Southwestern Medical Center for sharing the murine INS-1 cells and the insulin detection methods, and to Dr. Jerry Shay for providing the Lentiviral construct of CHGB shRNA. We thank Dr. Mahesh Chandak and Ms. Sutonuka Bhar (then at the University of Florida) for their technical assistance, and Prof. Tsan Sam Xiao at Case Western Reserve University for his advice and assistance on modeling from the cryo-EM maps. Special thanks go to Drs. Thomas Klose and Wen Jiang at the Purdue University, Drs. Ken Taylor, Xiaofeng Zheng, Nilakshee Battacharya and others at Florida State University, Drs. Ulrich Baxa, Thomas Edwards, Adam Wier and others at the National Cryo-EM Facility (NCEF) of National Cancer Institute (NCI) for their technical support in data collection in their microscopes. Isolated rat islets for high pressure freezing experiments were kindly provided by Prof. Michele Solimena at Technical University Dresden, Germany (to the Verkade lab). Some of the bilayer experiments were performed in a rig assembled in Dr. Feng Qin{\textquoteright}s laboratory in the University at Buffalo. We thank Drs. Jerry Shay (UT Southwestern), Ken Taylor (Florida State University), Wen Jiang (Purdue University) and Sixue Chen (University of Florida) for working with Q-XJ on joint grants related to this work. Funding Information: The work in the Jiang lab was supported by NIH (R21GM131231, R01GM111367, and R01GM093271 to Q-XJ), CF Foundation (JIANG15G0 to Q-XJ), Welch Foundation (I-1684 to Q-XJ) and CPRIT (RP120474 to Q-XJ), and by an AHA National Innovative Award (12IRG9400019 to Q-XJ), an NIGMS EUREKA Award (R01GM088745 to Q-XJ), a pilot grant from the Office of Research at the University of Florida (to Q-XJ), and startup funds from the University of Texas Southwestern Medical Center, the University of Florida and the Hauptman-Woodward Medical Research Institute. Some of the experiments reported here were performed in a laboratory constructed with support from NIH (C06RR30414). Besides EM facilities at home institutions, the cryo-EM studies in the Jiang lab were supported by SEM4 consortium at the Florida State University (grant #1U24GM116788) with Q-XJ as one of the MPIs, the National Cancer Institute{\textquoteright}s National Cryo-EM Facility (NCEF) at the Frederick National Laboratory for Cancer Research under a contract HSSN261200800001E, the NIH-funded consortium at the Purdue University (U24GM116789) with Q-XJ as one of the co-PIs as well as facilities at the Case Western Reserve University and the New York Structural Biology Center under the support of the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24GM129539) and by grants from the Simons Foundation (SF349247) and NY State Assembly Majority. Publisher Copyright: Copyright {\textcopyright} 2023 Yadav, Wang, Ouwendijk, Cross, Wang, Qin, Verkade, Zhu and Jiang.",

year = "2023",

month = jun,

day = "26",

doi = "10.3389/fnmol.2023.1205516",

language = "English",

volume = "16",

journal = "Frontiers in Molecular Neuroscience",

issn = "1662-5099",

publisher = "Frontiers Media S.A.",

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TY - JOUR

T1 - Chromogranin B (CHGB) is dimorphic and responsible for dominant anion channels delivered to cell surface via regulated secretion

AU - Yadav, Gaya P.

AU - Wang, Haiyuan

AU - Ouwendijk, Joke

AU - Cross, Stephen

AU - Wang, Qiaochu

AU - Qin, Feng

AU - Verkade, Paul

AU - Zhu, Michael X.

AU - Jiang, Qiu Xing

N1 - Funding Information: We are grateful to Dr. Barbara Ehrlich (Yale University) for sharing a construct for mouse CHGB, to Dr. Herbert Y. Gaisano (University of Toronto) for a syncollin-pHluorin construct, to Dr. Kuixing Zhang (UC San Diego) for sharing his techniques for releasing granular contents from endocrine cells, to Dr. Wen-Hong Li at UT Southwestern Medical Center for sharing the murine INS-1 cells and the insulin detection methods, and to Dr. Jerry Shay for providing the Lentiviral construct of CHGB shRNA. We thank Dr. Mahesh Chandak and Ms. Sutonuka Bhar (then at the University of Florida) for their technical assistance, and Prof. Tsan Sam Xiao at Case Western Reserve University for his advice and assistance on modeling from the cryo-EM maps. Special thanks go to Drs. Thomas Klose and Wen Jiang at the Purdue University, Drs. Ken Taylor, Xiaofeng Zheng, Nilakshee Battacharya and others at Florida State University, Drs. Ulrich Baxa, Thomas Edwards, Adam Wier and others at the National Cryo-EM Facility (NCEF) of National Cancer Institute (NCI) for their technical support in data collection in their microscopes. Isolated rat islets for high pressure freezing experiments were kindly provided by Prof. Michele Solimena at Technical University Dresden, Germany (to the Verkade lab). Some of the bilayer experiments were performed in a rig assembled in Dr. Feng Qin’s laboratory in the University at Buffalo. We thank Drs. Jerry Shay (UT Southwestern), Ken Taylor (Florida State University), Wen Jiang (Purdue University) and Sixue Chen (University of Florida) for working with Q-XJ on joint grants related to this work. Funding Information: The work in the Jiang lab was supported by NIH (R21GM131231, R01GM111367, and R01GM093271 to Q-XJ), CF Foundation (JIANG15G0 to Q-XJ), Welch Foundation (I-1684 to Q-XJ) and CPRIT (RP120474 to Q-XJ), and by an AHA National Innovative Award (12IRG9400019 to Q-XJ), an NIGMS EUREKA Award (R01GM088745 to Q-XJ), a pilot grant from the Office of Research at the University of Florida (to Q-XJ), and startup funds from the University of Texas Southwestern Medical Center, the University of Florida and the Hauptman-Woodward Medical Research Institute. Some of the experiments reported here were performed in a laboratory constructed with support from NIH (C06RR30414). Besides EM facilities at home institutions, the cryo-EM studies in the Jiang lab were supported by SEM4 consortium at the Florida State University (grant #1U24GM116788) with Q-XJ as one of the MPIs, the National Cancer Institute’s National Cryo-EM Facility (NCEF) at the Frederick National Laboratory for Cancer Research under a contract HSSN261200800001E, the NIH-funded consortium at the Purdue University (U24GM116789) with Q-XJ as one of the co-PIs as well as facilities at the Case Western Reserve University and the New York Structural Biology Center under the support of the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24GM129539) and by grants from the Simons Foundation (SF349247) and NY State Assembly Majority. Publisher Copyright: Copyright © 2023 Yadav, Wang, Ouwendijk, Cross, Wang, Qin, Verkade, Zhu and Jiang.

PY - 2023/6/26

Y1 - 2023/6/26

N2 - Regulated secretion is conserved in all eukaryotes. In vertebrates granin family proteins function in all key steps of regulated secretion. Phase separation and amyloid-based storage of proteins and small molecules in secretory granules require ion homeostasis to maintain their steady states, and thus need ion conductances in granule membranes. But granular ion channels are still elusive. Here we show that granule exocytosis in neuroendocrine cells delivers to cell surface dominant anion channels, to which chromogranin B (CHGB) is critical. Biochemical fractionation shows that native CHGB distributes nearly equally in soluble and membrane-bound forms, and both reconstitute highly selective anion channels in membrane. Confocal imaging resolves granular membrane components including proton pumps and CHGB in puncta on the cell surface after stimulated exocytosis. High pressure freezing immuno-EM reveals a major fraction of CHGB at granule membranes in rat pancreatic β-cells. A cryo-EM structure of bCHGB dimer of a nominal 3.5 Å resolution delineates a central pore with end openings, physically sufficient for membrane-spanning and large single channel conductance. Together our data support that CHGB-containing (CHGB+) channels are characteristic of regulated secretion, and function in granule ion homeostasis near the plasma membrane or possibly in other intracellular processes.

AB - Regulated secretion is conserved in all eukaryotes. In vertebrates granin family proteins function in all key steps of regulated secretion. Phase separation and amyloid-based storage of proteins and small molecules in secretory granules require ion homeostasis to maintain their steady states, and thus need ion conductances in granule membranes. But granular ion channels are still elusive. Here we show that granule exocytosis in neuroendocrine cells delivers to cell surface dominant anion channels, to which chromogranin B (CHGB) is critical. Biochemical fractionation shows that native CHGB distributes nearly equally in soluble and membrane-bound forms, and both reconstitute highly selective anion channels in membrane. Confocal imaging resolves granular membrane components including proton pumps and CHGB in puncta on the cell surface after stimulated exocytosis. High pressure freezing immuno-EM reveals a major fraction of CHGB at granule membranes in rat pancreatic β-cells. A cryo-EM structure of bCHGB dimer of a nominal 3.5 Å resolution delineates a central pore with end openings, physically sufficient for membrane-spanning and large single channel conductance. Together our data support that CHGB-containing (CHGB+) channels are characteristic of regulated secretion, and function in granule ion homeostasis near the plasma membrane or possibly in other intracellular processes.

KW - cell surface anion channels

KW - cryo-EM structures

KW - dimorphic CHGB

KW - regulated secretory pathways

KW - secretory granule exocytosis

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U2 - 10.3389/fnmol.2023.1205516

DO - 10.3389/fnmol.2023.1205516

M3 - Article (Academic Journal)

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JO - Frontiers in Molecular Neuroscience

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M1 - 1205516

ER -

Chromogranin B (CHGB) is dimorphic and responsible for dominant anion channels delivered to cell surface via regulated secretion

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