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Calcium Sensor Regulation of the Cav2.1 Ca2+ Channel Contributes

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Calcium Sensor Regulation of the Cav2.1 Ca2+ Channel Contributes 2+ Calcium sensor regulation of the CaV2.1 Ca channel contributes to long-term potentiation and spatial learning Evanthia Nanoua, Todd Scheuera, and William A. Catteralla,1 aDepartment of Pharmacology, University of Washington, Seattle, WA 98195-7280 Contributed by William A. Catterall, September 29, 2016 (sent for review September 2, 2016; reviewed by Diane Lipscombe and Gerald W. Zamponi) Many forms of short-term synaptic plasticity rely on regulation of Presynaptic signals may also contribute to this series of events by 2+ presynaptic voltage-gated Ca type 2.1 (CaV2.1) channels. How- strengthening glutamatergic transmission at hippocampal syn- ever, the contribution of regulation of CaV2.1 channels to other apses (20); however, at the outset of these experiments, we did forms of neuroplasticity and to learning and memory are not not expect that changes in short-term synaptic plasticity would known. Here we have studied mice with a mutation (IM-AA) that have major effects on LTP. To further explore the role of Ca 2.1 channel regulation by disrupts regulation of CaV2.1 channels by calmodulin and related V calcium sensor proteins. Surprisingly, we find that long-term po- CaS proteins in spatial learning and memory, we used knockin tentiation (LTP) of synaptic transmission at the Schaffer collateral- mice with paired alanine substitutions for the isoleucine and CA1 synapse in the hippocampus is substantially weakened, even methionine residues in the IQ-like motif (IM-AA) in their though this form of synaptic plasticity is thought to be primarily C-terminal domain (21), and we studied LTP and spatial learning and memory in IM-AA mice. In addition to the previously docu- generated postsynaptically. LTP in response to θ-burst stimulation mented alterations in short-term facilitation in Schaffer collateral and to 100-Hz tetanic stimulation is much reduced. However, a (SC)-CA1 synapses in acute hippocampal slices (21), we found normal level of LTP can be generated by repetitive 100-Hz stimu- major deficits in LTP and in spatial learning and memory, which lation or by depolarization of the postsynaptic cell to prevent 2+ reveal unexpected connections among presynaptic neuroplasticity, block of NMDA-specific glutamate receptors by Mg . The ratio postsynaptic LTP, and spatial learning and memory. NEUROSCIENCE of postsynaptic responses of NMDA-specific glutamate receptors to those of AMPA-specific glutamate receptors is decreased, but Results the postsynaptic current from activation of NMDA-specific gluta- IM-AA Mutation in CaV2.1 Channels Impairs LTP of SC-CA1 Pyramidal mate receptors is progressively increased during trains of stimuli Cell Synapses. In the acute hippocampal slice preparation, re- and exceeds WT by the end of 1-s trains. Strikingly, these impair- petitive bursts of stimulation at the frequency of θ-waves elicit ments in long-term synaptic plasticity and the previously documented LTP in response to a natural stimulus pattern (22, 23). We ex- impairments in short-term synaptic plasticity in IM-AA mice are asso- amined whether IM-AA synapses might have changes in long- ciated with pronounced deficits in spatial learning and memory in term synaptic modulation induced by θ-burst stimulation (TBS) context-dependent fear conditioning and in the Barnes circular maze. (Materials and Methods). We found that LTP, induced by TBS of Thus, regulation of CaV2.1 channels by calcium sensor proteins is re- SC fibers while holding the membrane potential of postsynaptic quired for normal short-term synaptic plasticity, LTP, and spatial CA1 neurons at −40 mV, was significantly reduced in IM-AA learning and memory in mice. synapses compared with WT (Fig. 1A). These results indicate that regulation of CaV2.1 channels by CaS proteins contributes calcium channel | calmodulin | synaptic plasticity | calcium sensor to long-term synaptic plasticity as well as short-term plasticity. proteins | hippocampus Significance ctivity-dependent modification of synaptic strength in syn- Aapses in the central nervous system is important for hippocampal- Learning and memory are caused by changes in strength of dependent information processing and for spatial learning and communication between neurons at synapses. Both brief changes memory (1). Short-term and long-term modifications in synaptic (short-term plasticity) and long-lasting changes (long-term plas- strength are regulated by the frequency and pattern of presynaptic ticity) are important. Synaptic transmission is initiated by calcium + spiking (2–5). Regulation of voltage-gated Ca2 channel type 2.1 channels, which are regulated by calcium-sensor proteins that 2+ (CaV2.1) by calmodulin (CaM) and related Ca sensor (CaS) induce short-term synaptic plasticity. We studied genetically + proteins causes Ca2 -dependent facilitation and inactivation of P/Q- modified mice with a mutation in the binding site for calcium- + type Ca2 currents (6–12) that results in short-term facilitation and sensor proteins on calcium channels, which alters short-term rapid depression of synaptic transmission (9, 12–14). Deletion synaptic plasticity. Surprisingly, we found that synapses in the of the gene encoding CaV2.1 channels (15) or mutation of their hippocampus of these mice also have impaired long-term po- CaS protein binding domain (12–14) impairs short-term synaptic tentiation. In addition, these mutant mice have impaired spa- plasticity. Although regulation of CaV2.1 channels contributes to tial learning and memory. Our results show that disruption of short-term synaptic plasticity in multiple types of synapses, the calcium-channel regulation by calcium-sensor proteins alters functional role of this form of synaptic regulation in learning both short-term and long-term plasticity, and these changes and memory is unknown. impair spatial learning and memory. Long-term potentiation (LTP) of synaptic transmission in the hippocampus is thought to be important for spatial learning and Author contributions: E.N., T.S., and W.A.C. designed research; E.N. performed research; memory (16). High-frequency stimuli induce LTP, which depends E.N., T.S., and W.A.C. analyzed data; and E.N. and W.A.C. wrote the paper. + on postsynaptic Ca2 entry via NMDA-specific glutamate recep- Reviewers: D.L., Brown University; and G.W.Z., University of Calgary. tors and activation of calcium/calmodulin-dependent protein The authors declare no conflict of interest. kinase II (17, 18). These events cause increased activity of AMPA- 1To whom correspondence should be addressed. Email: [email protected]. specific glutamate receptors via both direct phosphorylation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and enhanced trafficking to the postsynaptic membrane (19). 1073/pnas.1616206113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1616206113 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 The response of NMDA receptors to glutamate is controlled A + 400 * by voltage-dependent Mg2 block of the pore. To determine TBS 400 + whether relief of Mg2 blockade of NMDA receptors would en- 300 300 hance LTP at IM-AA synapses, we induced LTP while depolarizing postsynaptic CA1 pyramidal neurons to 0 mV during the tetanus. 200 200 Under these conditions, there was no significant difference be- tween the potentiated EPSCs of WT and IM-AA synapses (Fig. EPSC (%) A 100 100 2 ). Furthermore, LTP comparable to WT could be elicited LTP (% baseline) LTP using a stronger, three-train stimulus paradigm in IM-AA syn- 0 apses held at −60 mV (Fig. 2B). Taken together, these results 20 40 60 80 demonstrate that the IM-AA mutation causes weakened LTP, Time (min) which can be restored by stronger activation of NMDA receptors by + B using depolarization to reverse Mg2 block (Fig. 2A)orbyapplying 400 400 *** TET additional tetanic stimuli (Fig. 2B). To better understand the basis for altered LTP induction, we 300 300 directly recorded the EPSC during the trains of stimuli used to 200 elicit LTP. Mean peak amplitudes of EPSCs during 100-Hz trains 200 in IM-AA synapses were slightly increased relative to WT synapses, EPSC (%) 100 and the integral of EPSCs was greater (Fig. S1). These EPSCs re- 100 flect primarily the contribution of AMPA receptors, which are LTP (% baseline) LTP 0 rapidly activated by released glutamate. To estimate the efficacy of postsynaptic depolarization to induce LTP more accurately, we 20 40 60 80 Time (min) C 400 400 D-AP5 A 400 TET+DEP 400 300 TET 300 300 300 200 200 (% baseline) EPSC (%) 200 200 100 100 LTP EPSC (%) 100 0 100 LTP (% baseline) LTP 20 40 60 80 0 Time (min) 20 40 60 80 Fig. 1. IM-AA mutation reduces LTP in SC-CA1 synapses. (A) LTP induced at Time (min) − θ B 40 mV by eight trains of -burst stimulation (10 bursts delivered at 5 Hz, 400 TET 400 each burst consisted of 10 pulses at 100 Hz) from WT (black, n = 6) and IM- = AA (red, n 7) (Left). LTP values from individual cells (open symbols) and 300 300 average LTP values (solid symbols) from WT (black) and IM-AA (red) (Right). (B) LTP induced by two 100-Hz trains at −60 mV in the presence of 1 μM 200 ω-Ctx, 50 μM picrotoxin, and 10 μM CGP55845 hydrochloride from WT (black, 200 = = n 8) and IM-AA (red, n 8) (Left). (Right) Summarized LTP values as in A. (% baseline) EPSC (%) (C) LTP induced by two 100-Hz trains at 0 mV in the presence of 1 μM ω-Ctx, 100 100 50 μM picrotoxin, and 10 μM CGP55845 hydrochloride from WT (black, n = 7) LTP and IM-AA (red, n = 6) (Left). (Right) Summarized LTP values as in A.*P < 0 0.05; ***P < 0.001. 20 40 60 80 Time (min) C We investigated whether reduced LTP in IM-AA SC-CA1 250 synapses was specific to the mode of induction of LTP by com- paring LTP in WT and IM-AA mice induced by tetanic stimu- 200 lation.
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