James B. Ames

N-methyl-D-aspartate (NMDA) receptors (NMDARs) are Ca²⁺-permeable ionotropic glutamate receptors in the brain that have critical roles in learning, memory, neural development, and synaptic plasticity. NMDARs are heterotetrameric Ca²⁺ channels, which open upon binding to the neurotransmitters, glutamate and glycine. Channel opening causes Ca²⁺ influx that activates a range of Ca²⁺-dependent cellular processes, including activation of Ca²⁺-dependent enzymes, which mediates various forms of synaptic plasticity. Prolonged channel opening elevates the intracellular Ca²⁺ level to a cytotoxic concentration. To maintain Ca²⁺ homeostasis, NMDAR channel activity is finely regulated by α-actinin (ACTN), which promotes channel opening by reducing the closed time, and by calmodulin (CaM), which promotes Ca²⁺-dependent channel desensitization (CDD). Defects in the regulation of NMDAR function are associated with a spectrum of neurological diseases. In this review, we integrate cryo-EM structures of NMDARs, NMR structures of the NMDAR cytosolic C0 domain of the GluN1 and GluN2A subunits bound to Ca²⁺-bound CaM (Ca²⁺-CaM), and various structures of α-actinin-1 (ACTN1) to construct structural models of NMDAR in the open channel state bound to Ca²⁺-free ACTN1 and the agonist-bound, desensitized channel state bound to Ca²⁺-CaM. These structural models provide insights into the Ca²⁺-dependent conformational changes that promote CDD.

The L-type voltage-gated Ca²⁺ channel (Ca_V1.2) controls gene expression, cardiac function, and neuronal excitability. Mutations in Ca_V1.2 that disrupt channel function are implicated in cardiac arrhythmias, vascular dysfunction, Timothy Syndrome, and epilepsy. Calcium-binding protein 1 (CaBP1) binds to the IQ-motif in Ca_V1.2 (residues 1640-1665), blocks Ca²⁺-dependent inactivation (CDI), and promotes Ca²⁺-dependent facilitation (CDF). CaBP1 is 56% identical in sequence to calmodulin (CaM), and both proteins bind competitively to the IQ-motif. Our binding studies reveal that Ca²⁺ binding to CaBP1 is enhanced more than 40-fold when CaBP1 is bound to the IQ peptide. Also, the IQ peptide binds to Ca²⁺-bound CaBP1 (dissociation constant of 45 ± 10 nM) with 100-fold higher affinity than IQ binding to Ca²⁺-free CaBP1. We present NMR structures of Ca²⁺-CaBP1 bound to the IQ peptide, which reveal CaBP1 residues (A107, F111, M128, L131, I144, and M165) that contact IQ residues (I1654, Y1657, and F1658). Also, IQ residue K1662 forms a salt bridge with CaBP1 residue D140, which may explain why a K1662 charge reversal mutation causes 4-fold weaker IQ binding to CaBP1. Electrophysiology studies suggest that CaBP1 acts to increase the Ca_V1.2 channel open probability (Po). We propose that Ca²⁺ binding to the third and fourth EF-hands of CaBP1 and the binding of Ca²⁺-bound CaBP1 to the IQ-motif are important for Ca_V1.2 channel activation.

The L-type voltage-gated Ca²⁺ channel (Ca_V1.2) controls gene expression, cardiac contraction, and neuronal activity. The C-terminal cytosolic region of the Ca_V1.2 alpha subunit (α1C) contains two domains known as proximal and distal C-terminal regulatory domains (PCRD and DCRD), which have been suggested to control Ca²⁺-dependent channel inactivation (CDI). Previous studies identified a salt bridge interaction between PCRD and DCRD that might be central to channel function. In this study, we expressed and purified recombinant constructs of PCRD (residues 1680-1750) and DCRD (residues 2035-2089) in E. coli for NMR structural analysis. PCRD and DCRD each exhibit backbone NMR chemical shifts consistent with a random coil and lack of tertiary structure. A disordered random coil structure may explain the absence of electron density for these domains in recent cryo-EM structures of Ca_V1.2. Despite this lack of structure, fluorescence polarization binding assays reveal PCRD and DCRD each bind to the Ca_V1.2 IQ-motif (residues 1644-1668) with dissociation constants of 1.5 ± 1 and 14 ± 5 μM, respectively. Both PCRD and DCRD also become insoluble in the presence of high micromolar levels of the IQ peptide, consistent with each domain forming an insoluble complex with the IQ peptide. AlphaFold3 predicts that DCRD adopts a 3-helix bundle that binds to the helical IQ-motif, while PCRD was previously suggested to form a 4-helix bundle. We propose that the PCRD and DCRD bind to opposite sides of the helical IQ-motif, which may oppose calmodulin (CaM) binding to Ca_V1.2 and thus modulate channel CDI.

N-methyl-D-aspartate receptors (NMDARs) consist of glycine-binding GluN1 and glutamate-binding GluN2 subunits that form tetrameric ion channels. NMDARs in the brain are important for controlling neuronal excitability to promote synaptic plasticity. The cytoskeletal protein, α-actinin-1 (100 kDa, called ACTN1) binds to the cytosolic C0 domain of GluN1 (residues 841–865) that may play a role in the Ca²⁺-dependent desensitization of NMDAR channels. Mutations that disrupt NMDAR channel function are linked to Alzheimer’s disease, depression, stroke, epilepsy, and schizophrenia. NMR chemical shift assignments are reported here for the C-terminal EF-hand domain of ACTN1 (residues 824–892, called ACTN_EF34) and ACTN_EF34 bound to the GluN1 C0 domain (BMRB numbers 52385 and 52386, respectively).

Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5^R22A) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5^WT). Here, we present ITC, NMR, and functional analysis of GCAP5^R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5^R22A binds a total of 3 Ca²⁺, including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5^WT were not detected by ITC, suggesting that R22A may affect the binding of Ca²⁺ to these sites. The NMR-derived structure of GCAP5^R22A is overall similar to that of GCAP5^WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5^R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5^R22A with ∼25% higher affinity compared to GCAP5^WT, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.

Retinal membrane guanylyl cyclases (RetGCs) in vertebrate rod and cone photoreceptors are activated by a family of neuronal Ca²⁺ sensor proteins called guanylyl cyclase activating proteins (GCAP1-7). GCAP5 from zebrafish photoreceptors binds to RetGC and confers Ca²⁺/Fe²⁺-dependent regulation of RetGC enzymatic activity that promotes the recovery phase of visual phototransduction. We report NMR chemical shift assignments of GCAP5 with a R22A mutation (called GCAP5^R22A) that abolishes protein dimerization and activates RetGC with 3-fold higher activity than that of wild type GCAP5 (BMRB No. 51,783).

N-methyl-D-aspartate receptors (NMDARs) consist of glycine-binding GluN1 and glutamate-binding GluN2 subunits that form tetrameric ion channels. NMDARs in the neuronal post-synaptic membrane are important for controlling neuroplasticity and synaptic transmission in the brain. Calmodulin (CaM) binds to the cytosolic C0 domains of both GluN1 (residues 841–865) and GluN2 (residues 1004–1024) that may play a role in the Ca²⁺-dependent desensitization of NMDAR channels. Mutations that disrupt Ca²⁺-dependent desensitization of NMDARs are linked to Alzheimer’s disease, depression, stroke, epilepsy, and schizophrenia. NMR chemical shift assignments are reported here for Ca²⁺-saturated CaM bound to the GluN2A C0 domain of NMDAR (BMRB no. 51821).

Neuroplasticity and synaptic transmission in the brain are regulated by N-methyl-D-aspartate receptors (NMDARs) that consist of hetero-tetrameric combinations of the glycine-binding GluN1 and glutamate-binding GluN2 subunits. Calmodulin (CaM) binds to the cytosolic C0 domain of GluN1 (residues 841–865) that may play a role in the Ca²⁺-dependent inactivation (CDI) of NMDAR channel activity. Dysregulation of NMDARs are linked to various neurological disorders, including Alzheimer’s disease, depression, stroke, epilepsy, and schizophrenia. Here, we report complete NMR chemical shift assignments of Ca²⁺-saturated CaM bound to the GluN1 C0 domain of the human NMDAR (BMRB no. 51715).

Retinal cyclic nucleotide-gated (CNG) ion channels bind to intracellular cGMP and mediate visual phototransduction in photoreceptor rod and cone cells. Retinal rod CNG channels form hetero-tetramers comprised of three CNGA1 and one CNGB1 protein subunits. Cone CNG channels are similar tetramers consisting of three CNGA3 and one CNGB3 subunits. Calmodulin (CaM) binds to two distinct sites (CaM1: residues 565–587 and CaM2: residues 1120–1147) within the cytosolic domains of rod CNGB1. The binding of Ca²⁺-bound CaM to CNGB1 promotes the Ca²⁺-induced desensitization of CNG channels in retinal rods that may be important for photoreceptor light adaptation. Mutations that affect Ca²⁺-dependent CNG channel function are responsible for inherited forms of blindness. In this review, we propose structural models of the rod CNG channel bound to CaM that suggest how CaM might cause channel desensitization and how dysregulation of the channel may lead to retinal disease.