TRPM7 is critical for short-term synaptic depression by regulating synaptic vesicle endocytosis
- Department of Biological Sciences, University of Illinois at Chicago, United States
- School of Life Science, Jiangxi Science & Technology Normal University, China
- Department of Chemistry, University of Illinois at Chicago, United States
- Department of Biological Sciences, University of North Texas at Dallas, United States
- Department of Anatomy and Cell Biology, University of Illinois at Chicago, United States
Figures
Figure 1 with 4 supplements
Transient receptor potential melastatin 7 (TRPM7) is important for endocytic fission during endocytosis in chromaffin cells.
(A) Representative endocytic events, as membrane conductance (Re), membrane capacitance (Im), and the fission-pore conductance (Gp), in wild-type (WT) and TRPM7 knockoout (KO) cells. (B–E) The …
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Figure 1—source data 1
TRPM7 and single vesicle endocytosis in chromaffin cells.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Transient receptor potential melastatin 7 (TRPM7) depletion in adrenal medulla and cerebral cortex of the brain and adrenal medulla.
(A–B) RT-PCR of total RNA from adrenal medulla (A) and cerebral cortex (B) showing presence or absence of exon 17 of trpm7 in the differentially sized amplicons.
Figure 1—figure supplement 2
Transient receptor potential melastatin 7 (TRPM7) is important for endocytic kinetics in chromaffin cells.
(A) Pie chart showing percentages of endocytic events with their fission-pore durations in three different ranges (<5 ms; >5 ms, and <15 ms; >15 ms) from wild-type (WT) (left) and knockout (KO) …
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Figure 1—figure supplement 2—source data 1
Increases in the fission-pore duration in KO cells.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig1-figsupp2-data1-v2.xlsx
Figure 1—figure supplement 3
Endocytosis-associated current drift is independent of the capacitance size of endocytic events.
Endocytosis-associated current drifts recorded at all positive membrane potentials from Figure 1G were pooled and categorized into two groups based on their capacitance size ( >1 fF vs. <1 fF). …
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Figure 1—figure supplement 3—source data 1
Endocytic vesicle size and endocytosis-associated current drift.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig1-figsupp3-data1-v2.xlsx
Figure 1—figure supplement 4
An ionic current is specifically associated with single vesicle endocytosis.
(A) Representative membrane capacitance (Im) and patch membrane current (Ipatch) traces for exocytosis or endocytosis at the indicated membrane potentials. Dashed lines in cyan represent fitting …
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Figure 1—figure supplement 4—source data 1
Ionic current drifts associated with endocytosis but not exocytosis.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig1-figsupp4-data1-v2.xlsx
Figure 2 with 1 supplement
No change in large dense-core vesicles (LDCVs) exocytosis in transient receptor potential melastatin 7 (TRPM7) knockout (KO) chromaffin cells detected by carbon fiber amperometry.
(A) Amperometrical detection of catecholamine release from wild-type (WT) (upper) and KO (lower) chromaffin cells stimulated with 90 mM KCl for 5 s. Each amperometrical spike represents release from …
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Figure 2—source data 1
No change in exocytosis in KO chromaffin cells.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
No change in exocytosis in transient receptor potential melastatin 7 (TRPM7) knockout (KO) chromaffin cells measured by whole-cell capacitance recordings.
(A) Changes in capacitance (Cm, upper trace) and Ca2+ current (I, lower trace) from representative wild-type (WT) (black) and TRPM7 KO (red) chromaffin cells were simultaneously recorded in response …
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Figure 2—figure supplement 1—source data 1
Whole cell capactiance recordings in WT and KO cells.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 6 supplements
Synaptic vesicle endocytosis is impaired in transient receptor potential melastatin 7 (TRPM7) knockout (KO) neurons using pHluorin-based optical imaging assays.
(A) Normalized fluorescence changes of synaptophysin-pHluorin (sypHy) signals in wild-type (WT) (n = 139) and KO (n = 127) neurons at 2 mM [Ca2+]e. (B) Bar graph comparing the endocytic time …
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Figure 3—source data 1
Defects in synaptic vesicle endocytosis in KO neurons.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Endocytic defects of synaptic vesicles in transient receptor potential melastatin 7 (TRPM7) knockout (KO) neurons at physiological temperature of 34°C.
(A) Normalized fluorescence changes of synaptophysin-pHluorin (sypHy) signals in response to 200 action potentials (APs) at 20 Hz stimulations in wild-type (WT) (n = 161) and KO (n = 134) neurons at …
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Figure 3—figure supplement 1—source data 1
No change in synaptic vesicle endocytosis in KO neurons at physiological temperature.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 2
Measurements of reacidification rate of newly endocytosed vesicles from single synaptic terminals.
Representative time courses of pHluorin signals of individual synaptic terminals in the absence (black) or presence (gray) of bafilomycin A1, a vacuolar H+-ATPase inhibitor. To measure …
Figure 3—figure supplement 3
No difference in expression levels of endocytic proteins between wild-type (WT) and transient receptor potential melastatin 7 (TRPM7) knockout (KO) chromaffin cells.
(A–D) Representative confocal images of WT and TRPM7 KO chromaffin cells immunostained with tyrosine hydroxylase (TH) and endocytic proteins, such as clathrin (A), dynamin 1 (B), AP2 (C), or …
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Figure 3—figure supplement 3—source data 1
No change in endocytic proteins expressions in TRPM7 KO chromaffin cells.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig3-figsupp3-data1-v2.xlsx
Figure 3—figure supplement 4
No change in expression levels of endocytic proteins in presynaptic terminals of transient receptor potential melastatin 7 (TRPM7) knockout (KO) neurons.
(A, C, E, and G) Representative confocal images of presynaptic terminals from wild-type (WT) and TRPM7 KO neurons immunostained with endocytic proteins, such as clathrin (A), dynamin 1 (C), AP2 (E), …
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Figure 3—figure supplement 4—source data 1
No change in endocytic proteins expressions in presynaptic terminals of KO neurons.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig3-figsupp4-data1-v2.xlsx
Figure 3—figure supplement 5
Transient receptor potential melastatin 7 (TRPM7) may not be involved in synaptic vesicle exocytosis in inhibitory neurons.
(A–B) Representative (A) and quantification (B) of inhibitory postsynaptic currents (IPSCs) evoked by a single stimulation in wild-type (WT) and TRPM7 knockout (KO) neurons (WT: n = 14; KO: n = 13). …
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Figure 3—figure supplement 5—source data 1
No change in evoked IPSCs in TRPM7 neurons.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig3-figsupp5-data1-v2.xlsx
Figure 3—figure supplement 6
No difference in release probability of RRP vesicles between wild-type (WT) and transient receptor potential melastatin 7 (TRPM7) knockout (KO) neurons as measured from vGlut1-pHluorin experiments.
Release probability from individual terminals is calculated as the ratio of an averaged response induced by three single action potentials (APs) with 20 s intervals to RRP response induced by 40 APs …
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Figure 3—figure supplement 6—source data 1
No difference in release probability between WT and KO neurons.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig3-figsupp6-data1-v2.xlsx
Figure 4 with 2 supplements
Transient receptor potential melastatin 7 (TRPM7) as an ion channel is critical for endocytosis in both chromaffin cells and neurons.
(A) Averaged whole-cell TRPM7 currents, recorded from HEK 293 cells transduced with lentivirus encoding TRPM7WT or TRPM7LCF, in response to voltage ramps from –100 to +100 mV. (B) Quantifications …
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Figure 4—source data 1
Lentivirus-mediated TRPM7 versions in HEK 293 cells.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Confocal images for HEK 293 cells infected with lentivirus encoding with either empty vector, TRPM7WT or TRPM7LCF.
(A–C) Representative confocal images of HEK 293 cells infected with lentivirus encoding empty vector (A), TRPM7WT (B) or TRPM7LCF, (C) are on the left, phase contrast images of the same regions of …
Figure 4—figure supplement 2
Similar expression levels of TRPM7WT and TRPM7LCF in knockout (KO) chromaffin cells.
(A) Representative images of transient receptor potential melastatin 7 (TRPM7) KO cells, which were infected with lentivirus encoding empty vector (left), pCDH-EF1-flag-TRPM7WT (middle), or …
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Figure 4—figure supplement 2—source data 1
Lentivirus-mediated TRPM7 versions in chromaffin cells.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig4-figsupp2-data1-v2.xlsx
Figure 5 with 3 supplements
Transient receptor potential melastatin 7 (TRPM7) is important for presynaptic Ca2+ increase upon a train of stimulations.
(A) Normalized fluorescence changes of synaptophysin-pHluorin (sypHy) signal in wild-type (WT) (2 mM: n = 131 buttons; 0 mM: n = 68 buttons) and TRPM7 knockout (KO) (2 mM: n = 116 buttons; 0 mM: n = …
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Figure 5—source data 1
Decreased presynaptic Ca2+ signals during stimulation train.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
No difference in the resting Ca2+ concentration between wild-type (WT) and transient receptor potential melastatin 7 (TRPM7) knockout (KO) chromaffin cells.
(A) Fluorescence images of representative chromaffin cells loaded with Fura-2 AM from WT (top) and KO chromaffin cells (bottom). (B) Quantification of the ratio of fluorescence intensity excited at …
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Figure 5—figure supplement 1—source data 1
No difference in the resting Ca2+ concentration between WT and KO chromaffin cells.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig5-figsupp1-data1-v2.xlsx
Figure 5—figure supplement 2
No change in the basal Ca2+ concentration in presynaptic terminals of transient receptor potential melastatin 7 (TRPM7) knockout (KO) neurons.
(A) Fluorescence images of representative presynaptic terminals, identified with lentivirus-mediated vGlut1-pHluorin expression, from wild-type (WT) (top) and KO neurons (bottom) loaded with Fura-2 …
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Figure 5—figure supplement 2—source data 1
No difference in the resting Ca2+ concentration of synaptic terminals between WT and KO neurons.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig5-figsupp2-data1-v2.xlsx
Figure 5—figure supplement 3
The decay of Ca2+ signals after the cessation of the train stimulation is comparable between wild-type (WT) and transient receptor potential melastatin 7 (TRPM7) knockout (KO).
Decay of averaged SyGCaMP6f signals, elicited by a 100 action potential (AP) train at 20 Hz from each coverslip as shown in Figure 5C, was estimated by a single exponential fit from the first points …
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Figure 5—figure supplement 3—source data 1
Similar decay time constant of SyGCaMP6f siganls after stimulation train in WT and KO neurons.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig5-figsupp3-data1-v2.xlsx
Figure 6
Transient receptor potential melastatin 7 (TRPM7) is critical for presynaptic short-term depression in both inhibitory and excitatory synaptic transmissions.
(A–B) Representative traces of inhibitory postsynaptic currents (IPSCs) evoked by 250 stimulations at 10 Hz in wild-type (WT) (top in A) and TRPM7 knockout (KO) (bottom in A) neurons (A) and graph …
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Figure 6—source data 1
Enhanced short-term depression in KO neurons.
- https://cdn.elifesciences.org/articles/66709/elife-66709-fig6-data1-v2.xlsx
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | TRPM7fl/fl | Jackson LaboratoryStock No: 018784 | RRID:IMSR_JAX:018784 | |
| Strain, strain background (Mus musculus) | Nestin-Cre | Jackson LaboratoryStock No: 003771 | RRID:IMSR_JAX:003771 | |
| Strain, strain background (Mus musculus) | TH-Cre | Jackson LaboratoryStock No: 008601 | RRID:IMSR_JAX:008601 | |
| Cell line (human) | HEK 293FT | Thermo Fisher Scientific | Catalog number: R70007 | |
| Chemical compound, drug | CNQX | Tocris Bioscience | Cat# 0190 | |
| Chemical compound, drug | AP5 | Tocris Bioscience | Cat# 0106 | |
| Chemical compound, drug | TTX | Tocris Bioscience | Cat# 1078 | |
| Chemical compound, drug | Fura-2 AM | Thermo Fisher Scientific | Cat# F1221 | |
| Antibody | Anti-FLAG(Mouse monoclone M2) | Sigma-Aldrich | Cat# F1804RRID:AB_262044 | ICC (1:1000) |
| Antibody | Anti-TH(Rabbit polyclonal) | Abcam | Cat# ab6211RRID:AB_2240393 | ICC (1:2000) |
| Antibody | Anti-synaptophysin (Rabbit polyclonal) | Aviva Systems Biology | Cat# ARP45435_P050 RRID: AB_2048301 | ICC (1:1000) |
| Antibody | Anti-synaptophysin (Mouse monoclonal) | Sysy | Cat# 101011RRID: AB_887824 | ICC(1:1000) |
| Antibody | Anti-AP2(Mouse monoclonal) | abcam | Cat# ab2730RRID: AB_303255 | ICC(1:200) |
| Antibody | Aanti-Syt1(Mouse monoclonal) | Sysy | Cat# 105011RRID: AB_887832 | ICC(1:1000) |
| Antibody | Anti-clathrin(Mouse monoclonal) | Pierce | Cat# MA1-065RRID: AB_2083179 | ICC(1:500) |
| Antibody | Anti-dynamin(Rabbit polyclonal) | Abcam | Cat# ab3456RRID: AB_303818 | ICC (1:1000) |
| Antibody | Anti-mouse 555(Donkey polyclonal) | Thermo Fisher Scientific | Cat# A-31570RRID:AB_2536180 | ICC(1:1000) |
| Antibody | Anti-rabbit 488(Donkey polyclonal) | Thermo Fisher Scientific | Cat# A-21206 RRID:AB_2535792 | ICC(1:1000) |
| Antibody | Anti-mouse 647(Donkey polyclonal) | Thermo Fisher Scientific | Cat# A32787 RRID:AB_2762830 | ICC(1:1000) |
| Antibody | Anti-rabbit 555(Goat polyclonal) | Thermo Fisher Scientific | Cat# A32732 RRID:AB_2633281 | ICC(1:1000) |
| Antibody | Anti-mouse 488(Goat polyclonal) | Thermo Fisher Scientific | Cat# A32723 RRID:AB_2633275 | ICC(1:1000) |
| Antibody | Anti-rabbit 647(Goat polyclonal) | Thermo Fisher Scientific | Cat# A32733TR RRID:AB_2866492 | ICC(1:1000) |
| Recombinant DNA reagent | pCDH-EF1-MCS-T2A-copGFP | System Biosciences | Cat# CD526A-1 | A lentiviral vector |
| Recombinant DNA reagent | CMV::SypHy 4 A | Dr Leon LagnadoPubMed 16982422 | Cat# 24478RRID:Addgene_24478 | SypHy was subcloned into a pCDH lentiviral vector containing a synapsin 1 promoter in lab |
| Recombinant DNA reagent | pCDH-SYN1-sypHy | This paper | N/A | Plasmid was packaged into lentivirus, which was used to transduce primary neurons |
| Recombinant DNA reagent | pCDH-EF1-sypHy-T2A-TRPM7WT | This paper | N/A | Plasmid was used to transfect TRPM7 KO neurons |
| Recombinant DNA reagent | pCDH-EF1-sypHy-T2A-TRPM7LCF | This paper | N/A | Plasmid was used to transfect TRPM7 KO neurons |
| Recombinant DNA reagent | pCDH-EF1- TRPM7WT (Flag tag on N-term) | This paper | N/A | Plasmid was packaged into lentivirus, which was then used to infect TRPM7 KO chromaffin cells |
| Recombinant DNA reagent | pCDH-EF1-TRPM7LCF (Flag tag on N-term) | This paper | N/A | Plasmid was packaged into lentivirus, which was then used to infect TRPM7 KO chromaffin cells |
| Recombinant DNA reagent | TRPM7 (Flag tag on N-term) | Dr AM ScharenbergPubMed11385574 | Cat# 45482, AddgeneRRID:Addgene_45482 | DNA was subcloned into a pCDH lentiviral vector containing a EF1 promoter in lab |
| Recombinant DNA reagent | vGlut1-pHluorin | Dr V HauckePMID:21808019 | N/A | DNA was subcloned into a pCDH lentiviral vector containing a synapsin one promoter in lab |
| Recombinant DNA reagent | CaMKIIα-iGluSnFR | Dr ER ChapmanPMID:32515733 | N/A | S72A variant was used in this paper; DNA was subcloned into a pCDH lentiviral vector containing a synapsin 1 promoter in lab |
| Recombinant DNA reagent | Syn-GCaMP6f | Dr TA Ryan lab PMID:28162809 | N/A | DNA was subcloned into a pCDH lentiviral vector containing a synapsin 1 promoter in lab |
| Software, algorithm | Igor Pro 8 | WaveMetrics | RRID:SCR_000325 | |
| Software, algorithm | Excel | Microsoft | RRID:SCR_016137 | |
| Software, algorithm | Micro-Manager | ImageJ | RRID:SCR_000415 | |
| software, algorithm | FIJI | Fiji.sc | RRID:SCR_002285 | |
| Software, algorithm | Adobe Illustrator CS2 | Adobe | RRID:SCR_010279 | |
| Software, algorithm | Prism | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | NIS-Elements | Nikon | RRID:SCR_014329 |
