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Localized Calcium Signaling and the Control of Coupling at Cx36 Gap Junctions

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Localized Calcium Signaling and the Control of Coupling at Cx36 Gap Junctions Research Article: New Research | Novel Tools and Methods Localized calcium signaling and the control of coupling at Cx36 gap junctions https://doi.org/10.1523/ENEURO.0445-19.2020 Cite as: eNeuro 2020; 10.1523/ENEURO.0445-19.2020 Received: 25 October 2019 Revised: 29 January 2020 Accepted: 21 February 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 Moore et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 Localized calcium signaling and the control of coupling at Cx36 gap junctions 2 3 Abbreviated title: Calcium signaling at Cx36 gap junctions 4 5 Keith B. Moore1,†, Cheryl K. Mitchell1, Ya-Ping Lin1, Yuan-Hao Lee1, Eyad Shihabeddin1,2, and 6 John O'Brien1,2,* 7 8 1. Richard S. Ruiz, M.D. Department of Ophthalmology & Visual Science, McGovern Medical 9 School, The University of Texas Health Science Center at Houston, Houston, Texas, USA. 10 2. The MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, 11 Houston, Texas, USA. 12 †. Current Address: School of Public Health, The University of Texas Health Science Center at 13 Houston, Houston, Texas, USA. 14 15 Author contributions: KBM designed research, performed research, analyzed data, wrote the 16 paper; CKM performed research, analyzed data; YL performed research; Y-HL performed 17 research; ES analyzed data; JO designed research, analyzed data, wrote the paper 18 19 *Corresponding Author: John O’Brien 20 McGovern Medical School 21 6431 Fannin St., MSB 7.024, Houston, TX 77030 22 [email protected] 23 8 Figures 248 words in Abstract 24 3 tables 84 words in Significance statement 25 368 words in Introduction 26 1313 words in Discussion 27 28 Acknowledgements: The authors wish to thank Dr. Alejandro Vila for assistance with confocal 29 microscopy. 30 31 Conflicts of Interest: The authors declare no conflicts of interest. 32 33 Funding sources: This research was supported by NIH grant R01 EY012857 (JO), core grant P30 34 EY028102, and the Louisa Stude Sarofim endowment (JO). KBM was supported by training 35 grant T32 EY007024. 36 1 37 Abstract 38 A variety of electrical synapses are capable of activity-dependent plasticity, including both 39 activity-dependent potentiation and activity-dependent depression. In several types of 40 neurons, activity-dependent electrical synapse plasticity depends on changes in the local Ca2+ 41 environment. To enable study of local Ca2+ signaling that regulates plasticity, we developed a 42 GCaMP Ca2+ biosensor fused to the electrical synapse protein Connexin 36. Cx36-GCaMP 43 transfected into mammalian cell cultures formed gap junctions at cell-cell boundaries and 44 supported Neurobiotin tracer coupling that was regulated by protein kinase A signaling in the 45 same way as Cx36. Cx36-GCaMP gap junctions robustly reported local Ca2+ increases in 46 response to addition of a Ca2+ ionophore with increases in fluorescence that recovered during 47 washout. Recovery was strongly dependent on Na+-Ca2+ exchange activity. In cells transfected 48 with NMDA receptor subunits, Cx36-GCaMP revealed transient and concentration-dependent 49 increases in local Ca2+ upon brief application of glutamate. In HeLa cells, glutamate application 50 increased Cx36-GCaMP tracer coupling through a mechanism that depended in part on Ca2+, 51 calmodulin-dependent protein kinase II activity. This potentiation of coupling did not require 52 exogenous expression of glutamate receptors, but could be accomplished by endogenously 53 expressed glutamate receptors with pharmacological characteristics reminiscent of NMDA and 54 Kainate receptors. Analysis of RNA Sequencing data from HeLa cells confirmed expression of 55 NMDA receptor subunits NR1, NR2C and NR3B. In summary, Cx36-GCaMP is an effective tool 56 to measure changes in the Ca2+ microenvironment around Cx36 gap junctions. Furthermore, 57 HeLa cells can serve as a model system to study glutamate receptor-driven potentiation of 58 electrical synapses. 2 59 60 Significance 61 We have developed a Connexin 36-GCaMP3 fusion construct that effectively reports the Ca2+ 62 microenvironment in the vicinity of Cx36 gap junctions. This tool will be valuable to investigate 63 the dynamic changes in Ca2+ that are responsible for some forms of electrical synapse plasticity. 64 Furthermore, we have discovered that a widely used model system for in vitro studies, the HeLa 65 cell, endogenously expresses glutamate receptors that effectively drive intracellular Ca2+, 66 calmodulin-dependent protein kinase II signaling. This signaling can be exploited in many types 67 of studies. 3 68 Introduction 69 Electrical synapses provide a means to organize neurons into networks from which high- 70 order activity emerges. Plasticity is a fundamental property of electrical synapses, altering the 71 strength of electrical communication between coupled neurons and potentially playing a large 72 role in regulation of network states (Coulon and Landisman, 2017). Electrical synapse plasticity 73 is also a critical element of microcircuit functions. For example, individual auditory afferent 74 terminals that form mixed chemical and electrical synapses onto Mauthner neurons display a 75 high degree of variability in both electrical and chemical synaptic strength (Smith and Pereda, 76 2003), influencing the effect of any one auditory neuron on Mauthner cell responses. Both 77 components can be modified by activity, resulting in either potentiation or depression of 78 individual elements, with interdependence of chemical and electrical synapse activity (Pereda 79 et al., 2003b; Smith and Pereda, 2003). 80 In the fish Mauthner neurons, high-frequency stimulation of the afferent nerve results 81 in potentiation of electrical synapses (Yang et al., 1990; Pereda and Faber, 1996). Activity- 82 dependent potentiation of electrical synapses has also been observed in mammalian AII 83 amacrine cells (Kothmann et al., 2012) and inferior olive neurons (Turecek et al., 2014). 84 Common among these neural networks is the reliance on activation of NMDA receptors, influx 85 of extracellular Ca2+ and activation of Ca2+, calmodulin-dependent protein kinase II (CaMKII) 86 activity (Pereda et al., 1998; Pereda et al., 2003a; Kothmann et al., 2012; Turecek et al., 2014) 87 to potentiate coupling. In retinal AII amacrine cells, the potentiation has been demonstrated to 88 result from CaMKII-dependent phosphorylation of Cx36 (Kothmann et al., 2012). Ca2+- 89 dependent depression of electrical synapses has also been reported, depending on NMDA 4 90 receptors in inferior olive neurons (Mathy et al., 2014) and voltage-dependent Ca2+ channels in 91 thalamic reticular neurons (Sevetson et al., 2017), suggesting that the control of coupling by 92 Ca2+ entry can be subtle. 93 Because of the central role of Ca2+ signaling in activity-dependent modulation of 94 electrical synapse strength, we wished to investigate the relationship between the Ca2+ 95 microenvironment around Cx36 gap junctions and their functional plasticity. To accomplish 96 this, we developed a Cx36 construct fused to the genetically-encoded Ca2+ biosensor GCaMP3 97 (Tian et al., 2009). This construct is an effective tool to examine Ca2+ signaling in the context of 98 gap junction functional plasticity. 99 100 Materials and Methods 101 Clones 102 EGFP-N1 vector was obtained from Clontech (Mountain View, CA). A plasmid expressing 103 GCaMP3 in the EGFP-N1 vector was a gift from Dr. Loren Looger (Addgene plasmid #22692) 104 (Tian et al., 2009). Mouse Connexin 36 (Cx36) cDNA was a gift from Dr. Muayyad Al-Ubaidi 105 (University of Oklahoma) (Al-Ubaidi et al., 2000). NMDA receptor open reading frame clones 106 were a gift from Dr. Vasanthi Jayarman (University of Texas Health Science Center at Houston). 107 These included NR1 in pcDNA 3.1 (NCBI accession# 57847), NR2A in pcDNA 3.1 (NCBI 108 accession# 286233), NR2B in pcDNA 3.1 (NCBI accession# NM_012574), and NR2C in pRK (NCBI 109 accession# NM_012575.3). 110 Unless otherwise indicated, restriction enzymes, DNA polymerases and enzymes used 111 for cloning were obtained from New England Biolabs (Ipswich, MA). To produce C-terminal 5 112 EGFP-tagged Cx36, the mouse Cx36 cDNA was amplified by PCR using Pfu Ultra DNA polymerase 113 (Agilent, Santa Clara, CA) with primers T7 and JOB 284 (all primer sequences are listed in Table 114 I), the latter of which eliminates the stop codon of Cx36 and adds a KpnI site in frame with the 115 EGFP-N1 vector. The PCR product was cloned into EcoRI and KpnI sites of the EGFP-N1 vector 116 by conventional cloning. A C-terminal GCaMP3-tagged Cx36 was produced in a similar fashion 117 using primers T7 and JOB 285, the latter of which eliminates the stop codon of Cx36 and adds a 118 BclI site. The PCR product was digested with SmaI and BclI and cloned into AfeI and BglII sites 119 of the GCaMP3 vector. Resulting clones were fully sequenced. Cx36-GCaMP is available 120 through Addgene (plasmid # 123604). 121 A series of clones was created using the PB513B-1 dual expression vector of the 122 Piggybac Transposon System (System Biosciences LLC, Palo Alto, CA). The vector was modified 123 to accept a second user-derived sequence by deleting the GFP open reading frame driven by 124 the EF1a promoter by digestion with Bsu36I and BgmBI and inserting a linker containing PacI 125 and SbfI restriction sites followed by an IRES sequence derived from the vector pIRES DsRedT3- 126 KR24 (Moshiri et al., 2008).
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