Evanthia Nanou

Calcium Channels and Short-term Synaptic Plasticity

Voltage-gated Ca2+ channels in presynaptic nerve terminals initiate neurotransmitter release in response to depolarization by action potentials from the nerve axon. The strength of synaptic transmission is dependent on the third to fourth power of Ca2+ entry, placing the Ca2+ channels in a unique position for regulation of synaptic strength. Short-term synaptic plasticity regulates the strength of neurotransmission through facilitation and depression on the millisecond time scale and plays a key role in encoding information in the nervous system. CaV2.1 channels are the major source of Ca2+ entry for neurotransmission in the central nervous system. They are tightly regulated by Ca2+, calmodulin, and related Ca2+ sensor proteins, which cause facilitation and inactivation of channel activity. Emerging evidence reviewed here points to this mode of regulation of CaV2.1 channels as a major contributor to short-term synaptic plasticity of neurotransmission and its diversity among synapses.

Calcium sensor regulation of the CaV2.1 Ca2+ channel contributes to short-term synaptic plasticity in hippocampal neurons

Significance Information processing in the brain is mediated through synaptic connections between neurons, where neurotransmitter molecules released from presynaptic nerve terminals stimulate postsynaptic cells. Strength of synaptic transmission is increased transiently by short-term synaptic facilitation in response to repeated stimulation of nerve fibers. Synaptic transmission is initiated by calcium influx through calcium channels in presynaptic nerve terminals, which are regulated by calcium sensor proteins. We found that genetically modified mice in which we introduced a mutation in the binding site for calcium sensor proteins in presynaptic calcium channels have substantially altered facilitation in hippocampal synapses in response to pairs or trains of repetitive high-frequency stimuli. Our results show that disruption of calcium channel regulation by calcium sensor proteins impairs short-term facilitation in native synapses. Short-term synaptic plasticity is induced by calcium (Ca2+) accumulating in presynaptic nerve terminals during repetitive action potentials. Regulation of voltage-gated CaV2.1 Ca2+ channels by Ca2+ sensor proteins induces facilitation of Ca2+ currents and synaptic facilitation in cultured neurons expressing exogenous CaV2.1 channels. However, it is unknown whether this mechanism contributes to facilitation in native synapses. We introduced the IM-AA mutation into the IQ-like motif (IM) of the Ca2+ sensor binding site. This mutation does not alter voltage dependence or kinetics of CaV2.1 currents, or frequency or amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs); however, synaptic facilitation is completely blocked in excitatory glutamatergic synapses in hippocampal autaptic cultures. In acutely prepared hippocampal slices, frequency and amplitude of mEPSCs and amplitudes of evoked EPSCs are unaltered. In contrast, short-term synaptic facilitation in response to paired stimuli is reduced by ∼50%. In the presence of EGTA-AM to prevent global increases in free Ca2+, the IM-AA mutation completely blocks short-term synaptic facilitation, indicating that synaptic facilitation by brief, local increases in Ca2+ is dependent upon regulation of CaV2.1 channels by Ca2+ sensor proteins. In response to trains of action potentials, synaptic facilitation is reduced in IM-AA synapses in initial stimuli, consistent with results of paired-pulse experiments; however, synaptic depression is also delayed, resulting in sustained increases in amplitudes of later EPSCs during trains of 10 stimuli at 10–20 Hz. Evidently, regulation of CaV2.1 channels by CaS proteins is required for normal short-term plasticity and normal encoding of information in native hippocampal synapses.

Phosphorylation of Ser1928 mediates the enhanced activity of the L-type Ca2+ channel Cav1.2 by the β2-adrenergic receptor in neurons

β-Adrenergic regulation of the L-type calcium channel Cav1.2 exhibits surprising differences in the heart and brain. How adrenaline activates Cav1.2 The L-type Ca2+ channel Cav1.2 controls heart rate and neuronal excitability. Qian et al. found that enhancement of Cav1.2 channel activity in the brain by β-adrenergic receptor (βAR) signaling required phosphorylation of Ser1928, whereas in the heart, this site was dispensable for βAR-mediated regulation. In contrast to those from wild-type mice, hippocampal neurons from mice, in which Ser1928 of Cav1.2 was mutated to alanine, did not exhibit increased L-type calcium channel activity in response to β-adrenergic stimulation. Phosphorylation of Ser1928 involved signaling through the β2AR, but not through the β1AR, and this phosphorylation event enabled a particular form of long-term potentiation, a process linked to learning and memory. These results were in marked contrast to βAR-mediated regulation of Cav1.2 activity in cardiomyocytes, which involved β1AR and was independent of Ser1928. This differential regulation in the heart and brain implies that tissue-specific therapeutics could be identified. The L-type Ca2+ channel Cav1.2 controls multiple functions throughout the body including heart rate and neuronal excitability. It is a key mediator of fight-or-flight stress responses triggered by a signaling pathway involving β-adrenergic receptors (βARs), cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA). PKA readily phosphorylates Ser1928 in Cav1.2 in vitro and in vivo, including in rodents and humans. However, S1928A knock-in (KI) mice have normal PKA-mediated L-type channel regulation in the heart, indicating that Ser1928 is not required for regulation of cardiac Cav1.2 by PKA in this tissue. We report that augmentation of L-type currents by PKA in neurons was absent in S1928A KI mice. Furthermore, S1928A KI mice failed to induce long-term potentiation in response to prolonged theta-tetanus (PTT-LTP), a form of synaptic plasticity that requires Cav1.2 and enhancement of its activity by the β2-adrenergic receptor (β2AR)–cAMP–PKA cascade. Thus, there is an unexpected dichotomy in the control of Cav1.2 by PKA in cardiomyocytes and hippocampal neurons.

Modulation of CaV2.1 channels by neuronal calcium sensor-1 induces short-term synaptic facilitation

Facilitation and inactivation of P/Q-type Ca2+ currents mediated by Ca2+/calmodulin binding to Ca(V)2.1 channels contribute to facilitation and rapid depression of synaptic transmission, respectively. Other calcium sensor proteins displace calmodulin from its binding site and differentially modulate P/Q-type Ca2 + currents, resulting in diverse patterns of short-term synaptic plasticity. Neuronal calcium sensor-1 (NCS-1, frequenin) has been shown to enhance synaptic facilitation, but the underlying mechanism is unclear. We report here that NCS-1 directly interacts with IQ-like motif and calmodulin-binding domain in the C-terminal domain of Ca(V)2.1 channel. NCS-1 reduces Ca2 +-dependent inactivation of P/Q-type Ca2+ current through interaction with the IQ-like motif and calmodulin-binding domain without affecting peak current or activation kinetics. Expression of NCS-1 in presynaptic superior cervical ganglion neurons has no effect on synaptic transmission, eliminating effects of this calcium sensor protein on endogenous N-type Ca2+ currents and the endogenous neurotransmitter release machinery. However, in superior cervical ganglion neurons expressing wild-type Ca(V)2.1 channels, co-expression of NCS-1 induces facilitation of synaptic transmission in response to paired pulses and trains of depolarizing stimuli, and this effect is lost in Ca(V)2.1 channels with mutations in the IQ-like motif and calmodulin-binding domain. These results reveal that NCS-1 directly modulates Ca(V)2.1 channels to induce short-term synaptic facilitation and further demonstrate that CaS proteins are crucial in fine-tuning short-term synaptic plasticity.

Asynchronous Ca2+ current conducted by voltage-gated Ca2+ (CaV)-2.1 and CaV2.2 channels and its implications for asynchronous neurotransmitter release

We have identified an asynchronously activated Ca2+ current through voltage-gated Ca2+ (CaV)-2.1 and CaV2.2 channels, which conduct P/Q- and N-type Ca2+ currents that initiate neurotransmitter release. In nonneuronal cells expressing CaV2.1 or CaV2.2 channels and in hippocampal neurons, prolonged Ca2+ entry activates a Ca2+ current, IAsync, which is observed on repolarization and decays slowly with a half-time of 150–300 ms. IAsync is not observed after L-type Ca2+ currents of similar size conducted by CaV1.2 channels. IAsync is Ca2+-selective, and it is unaffected by changes in Na+, K+, Cl−, or H+ or by inhibitors of a broad range of ion channels. During trains of repetitive depolarizations, IAsync increases in a pulse-wise manner, providing Ca2+ entry that persists between depolarizations. In single-cultured hippocampal neurons, trains of depolarizations evoke excitatory postsynaptic currents that show facilitation followed by depression accompanied by asynchronous postsynaptic currents that increase steadily during the train in parallel with IAsync. IAsync is much larger for slowly inactivating CaV2.1 channels containing β2a-subunits than for rapidly inactivating channels containing β1b-subunits. IAsync requires global rises in intracellular Ca2+, because it is blocked when Ca2+ is chelated by 10 mM EGTA in the patch pipette. Neither mutations that prevent Ca2+ binding to calmodulin nor mutations that prevent calmodulin regulation of CaV2.1 block IAsync. The rise of IAsync during trains of stimuli, its decay after repolarization, its dependence on global increases of Ca2+, and its enhancement by β2a-subunits all resemble asynchronous release, suggesting that IAsync is a Ca2+ source for asynchronous neurotransmission.

Molecular Determinants of CaV2.1 Channel Regulation by Calcium-binding Protein-1

Background: Regulation of calcium channels by calcium-sensor proteins mediates short term synaptic plasticity. Results: Calcium-binding protein-1 (CaBP1) inhibits calcium channels through its N terminus and second EF-hand, which is inactive in calcium binding. Conclusion: Specific domains of CaBP1 are responsible for differential regulation, including an EF-hand inactive in calcium binding. Significance: These results reveal the molecular code used by calcium-sensor proteins to differentially regulate short term synaptic plasticity. Presynaptic CaV2.1 channels, which conduct P/Q-type Ca2+ currents, initiate synaptic transmission at most synapses in the central nervous system. Regulation of CaV2.1 channels by CaM contributes significantly to short term facilitation and rapid depression of synaptic transmission. Short term synaptic plasticity is diverse in form and function at different synapses, yet CaM is ubiquitously expressed. Differential regulation of CaV2.1 channels by CaM-like Ca2+ sensor (CaS) proteins differentially affects short term synaptic facilitation and rapid synaptic depression in transfected sympathetic neuron synapses. Here, we define the molecular determinants for differential regulation of CaV2.1 channels by the CaS protein calcium-binding protein-1 (CaBP1) by analysis of chimeras in which the unique structural domains of CaBP1 are inserted into CaM. Our results show that the N-terminal domain, including its myristoylation site, and the second EF-hand, which is inactive in Ca2+ binding, are the key molecular determinants of differential regulation of CaV2.1 channels by CaBP1. These findings give insight into the molecular code by which CaS proteins differentially regulate CaV2.1 channel function and provide diversity of form and function of short term synaptic plasticity.

Molecular determinants of modulation of Cav2.1 channels by visinin-like protein 2

Background: Regulation of calcium channels by calcium sensor proteins mediates short-term synaptic plasticity. Results: Visinin-like protein-2 (VILIP-2) increases facilitation of calcium channels through its N terminus, interlobe linker, and EF-hands 3 and 4. Conclusion: Specific domains of VILIP-2 are responsible for regulation, including adjacent EF-hands that bind calcium. Significance: These results reveal the molecular code used by calcium-sensor proteins to differentially regulate short-term synaptic plasticity. CaV2.1 channels, which conduct P/Q-type Ca2+ currents, initiate synaptic transmission at most synapses in the central nervous system. Ca2+/calmodulin-dependent facilitation and inactivation of these channels contributes to short-term facilitation and depression of synaptic transmission, respectively. Other calcium sensor proteins displace calmodulin (CaM) from its binding site, differentially regulate CaV2.1 channels, and contribute to the diversity of short-term synaptic plasticity. The neuronal calcium sensor protein visinin-like protein 2 (VILIP-2) inhibits inactivation and enhances facilitation of CaV2.1 channels. Here we examine the molecular determinants for differential regulation of CaV2.1 channels by VILIP-2 and CaM by construction and functional analysis of chimeras in which the functional domains of VILIP-2 are substituted in CaM. Our results show that the N-terminal domain, including its myristoylation site, the central α-helix, and the C-terminal lobe containing EF-hands 3 and 4 of VILIP-2 are sufficient to transfer its regulatory properties to CaM. This regulation by VILIP-2 requires binding to the IQ-like domain of CaV2.1 channels. Our results identify the essential molecular determinants of differential regulation of CaV2.1 channels by VILIP-2 and define the molecular code that these proteins use to control short-term synaptic plasticity.

Calcium sensor regulation of the CaV2.1 Ca2+ channel contributes to long-term potentiation and spatial learning

Significance Learning and memory are caused by changes in strength of communication between neurons at synapses. Both brief changes (short-term plasticity) and long-lasting changes (long-term plasticity) are important. Synaptic transmission is initiated by calcium channels, which are regulated by calcium-sensor proteins that induce short-term synaptic plasticity. We studied genetically modified mice with a mutation in the binding site for calcium-sensor proteins on calcium channels, which alters short-term synaptic plasticity. Surprisingly, we found that synapses in the hippocampus of these mice also have impaired long-term potentiation. In addition, these mutant mice have impaired spatial learning and memory. Our results show that disruption of calcium-channel regulation by calcium-sensor proteins alters both short-term and long-term plasticity, and these changes impair spatial learning and memory. Many forms of short-term synaptic plasticity rely on regulation of presynaptic voltage-gated Ca2+ type 2.1 (CaV2.1) channels. However, the contribution of regulation of CaV2.1 channels to other forms of neuroplasticity and to learning and memory are not known. Here we have studied mice with a mutation (IM-AA) that disrupts regulation of CaV2.1 channels by calmodulin and related calcium sensor proteins. Surprisingly, we find that long-term potentiation (LTP) of synaptic transmission at the Schaffer collateral-CA1 synapse in the hippocampus is substantially weakened, even though this form of synaptic plasticity is thought to be primarily generated postsynaptically. LTP in response to θ-burst stimulation and to 100-Hz tetanic stimulation is much reduced. However, a normal level of LTP can be generated by repetitive 100-Hz stimulation or by depolarization of the postsynaptic cell to prevent block of NMDA-specific glutamate receptors by Mg2+. The ratio of postsynaptic responses of NMDA-specific glutamate receptors to those of AMPA-specific glutamate receptors is decreased, but the postsynaptic current from activation of NMDA-specific glutamate receptors is progressively increased during trains of stimuli and exceeds WT by the end of 1-s trains. Strikingly, these impairments in long-term synaptic plasticity and the previously documented impairments in short-term synaptic plasticity in IM-AA mice are associated with pronounced deficits in spatial learning and memory in context-dependent fear conditioning and in the Barnes circular maze. Thus, regulation of CaV2.1 channels by calcium sensor proteins is required for normal short-term synaptic plasticity, LTP, and spatial learning and memory in mice.

Altered short-term synaptic plasticity and reduced muscle strength in mice with impaired regulation of presynaptic CaV2.1 Ca2+ channels

Significance Muscle contraction begins with release of neurotransmitter molecules from motor-nerve terminals onto muscle fibers. Strength of contraction depends on high-frequency stimulation of the nerve, which induces short-term increases in neurotransmitter release through presynaptic facilitation. Neurotransmitter release is initiated via calcium influx through voltage-gated calcium channels, which are regulated by calcium sensor (CaS) proteins. We found that genetically modified mice with a mutation in the binding site for CaS proteins on presynaptic calcium channels have reduced synaptic facilitation during high-frequency stimulation, resulting in reduced muscle strength, endurance, and coordination. Our results demonstrate critical roles for the regulation of calcium channels by CaS proteins in short-term presynaptic facilitation, with important consequences for motor control and muscle function in vivo. Facilitation and inactivation of P/Q-type calcium (Ca2+) currents through the regulation of voltage-gated Ca2+ (CaV) 2.1 channels by Ca2+ sensor (CaS) proteins contributes to the facilitation and rapid depression of synaptic transmission in cultured neurons that transiently express CaV2.1 channels. To examine the modulation of endogenous CaV2.1 channels by CaS proteins in native synapses, we introduced a mutation (IM-AA) into the CaS protein-binding site in the C-terminal domain of CaV2.1 channels in mice, and tested synaptic facilitation and depression in neuromuscular junction synapses that use exclusively CaV2.1 channels for Ca2+ entry that triggers synaptic transmission. Even though basal synaptic transmission was unaltered in the neuromuscular synapses in IM-AA mice, we found reduced short-term facilitation in response to paired stimuli at short interstimulus intervals in IM-AA synapses. In response to trains of action potentials, we found increased facilitation at lower frequencies (10–30 Hz) in IM-AA synapses accompanied by slowed synaptic depression, whereas synaptic facilitation was reduced at high stimulus frequencies (50–100 Hz) that would induce strong muscle contraction. As a consequence of altered regulation of CaV2.1 channels, the hindlimb tibialis anterior muscle in IM-AA mice exhibited reduced peak force in response to 50 Hz stimulation and increased muscle fatigue. The IM-AA mice also had impaired motor control, exercise capacity, and grip strength. Taken together, our results indicate that regulation of CaV2.1 channels by CaS proteins is essential for normal synaptic plasticity at the neuromuscular junction and for muscle strength, endurance, and motor coordination in mice in vivo.

Control of Excitation/Inhibition Balance in a Hippocampal Circuit by Calcium Sensor Protein Regulation of Presynaptic Calcium Channels

Activity-dependent regulation controls the balance of synaptic excitation to inhibition in neural circuits, and disruption of this regulation impairs learning and memory and causes many neurological disorders. The molecular mechanisms underlying short-term synaptic plasticity are incompletely understood, and their role in inhibitory synapses remains uncertain. Here we show that regulation of voltage-gated calcium (Ca2+) channel type 2.1 (CaV2.1) by neuronal Ca2+ sensor (CaS) proteins controls synaptic plasticity and excitation/inhibition balance in a hippocampal circuit. Prevention of CaS protein regulation by introducing the IM-AA mutation in CaV2.1 channels in male and female mice impairs short-term synaptic facilitation at excitatory synapses of CA3 pyramidal neurons onto parvalbumin (PV)-expressing basket cells. In sharp contrast, the IM-AA mutation abolishes rapid synaptic depression in the inhibitory synapses of PV basket cells onto CA1 pyramidal neurons. These results show that CaS protein regulation of facilitation and inactivation of CaV2.1 channels controls the direction of short-term plasticity at these two synapses. Deletion of the CaS protein CaBP1/caldendrin also blocks rapid depression at PV-CA1 synapses, implicating its upregulation of inactivation of CaV2.1 channels in control of short-term synaptic plasticity at this inhibitory synapse. Studies of local-circuit function revealed reduced inhibition of CA1 pyramidal neurons by the disynaptic pathway from CA3 pyramidal cells via PV basket cells and greatly increased excitation/inhibition ratio of the direct excitatory input versus indirect inhibitory input from CA3 pyramidal neurons to CA1 pyramidal neurons. This striking defect in local-circuit function may contribute to the dramatic impairment of spatial learning and memory in IM-AA mice. SIGNIFICANCE STATEMENT Many forms of short-term synaptic plasticity in neuronal circuits rely on regulation of presynaptic voltage-gated Ca2+ (CaV) channels. Regulation of CaV2.1 channels by neuronal calcium sensor (CaS) proteins controls short-term synaptic plasticity. Here we demonstrate a direct link between regulation of CaV2.1 channels and short-term synaptic plasticity in native hippocampal excitatory and inhibitory synapses. We also identify CaBP1/caldendrin as the calcium sensor interacting with CaV2.1 channels to mediate rapid synaptic depression in the inhibitory hippocampal synapses of parvalbumin-expressing basket cells to CA1 pyramidal cells. Disruption of this regulation causes altered short-term plasticity and impaired balance of hippocampal excitatory to inhibitory circuits.