Retinal guanylate cyclases (RetGCs) promote the Ca2+-dependent synthesis of cGMP that coordinates the recovery phase of visual phototransduction in retinal rods and cones. The Ca2+-sensitive activation of RetGCs is controlled by a family of photoreceptor Ca2+ binding proteins known as guanylate cyclase activator proteins (GCAPs). The Mg2+-bound/Ca2+-free GCAPs bind to RetGCs and activate cGMP synthesis (cyclase activity) at low cytosolic Ca2+ levels in light-activated photoreceptors. By contrast, Ca2+-bound GCAPs bind to RetGCs and inactivate cyclase activity at high cytosolic Ca2+ levels found in dark-adapted photoreceptors. Mutations in both RetGCs and GCAPs that disrupt the Ca2+-dependent cyclase activity are genetically linked to various retinal diseases known as cone-rod dystrophies. In this review, I will provide an overview of the known atomic-level structures of various GCAP proteins to understand how protein dimerization and Ca2+-dependent conformational changes in GCAPs control the cyclase activity of RetGCs. This review will also summarize recent structural studies on a GCAP homolog from zebrafish (GCAP5) that binds to Fe2+ and may serve as a Fe2+ sensor in photoreceptors. The GCAP structures reveal an exposed hydrophobic surface that controls both GCAP1 dimerization and RetGC binding. This exposed site could be targeted by therapeutics designed to inhibit the GCAP1 disease mutants, which may serve to mitigate the onset of retinal cone-rod dystrophies.

Neuronal calcium sensor (NCS) proteins are EF-hand containing Ca²⁺ binding proteins that regulate sensory signal transduction. Many NCS proteins (recoverin, GCAPs, neurocalcin and visinin-like protein 1 (VILIP1)) form functional dimers under physiological conditions. The dimeric NCS proteins have similar amino acid sequences (50% homology) but each bind to and regulate very different physiological targets. Retinal recoverin binds to rhodopsin kinase and promotes Ca²⁺-dependent desensitization of light-excited rhodopsin during visual phototransduction. The guanylyl cyclase activating proteins (GCAP1-5) each bind and activate retinal guanylyl cyclases (RetGCs) in light-adapted photoreceptors. VILIP1 binds to membrane targets that modulate neuronal secretion. Here, I review atomic-level structures of dimeric forms of recoverin, GCAPs and VILIP1. The distinct dimeric structures in each case suggest that NCS dimerization may play a role in modulating specific target recognition. The dimerization of recoverin and VILIP1 is Ca²⁺-dependent and enhances their membrane-targeting Ca²⁺-myristoyl switch function. The dimerization of GCAP1 and GCAP2 facilitate their binding to dimeric RetGCs and may allosterically control the Ca²⁺-dependent activation of RetGCs.

Retinal membrane guanylate cyclases (RetGC1 and RetGC2) are expressed in photoreceptor rod and cone cells, where they promote the onset of visual recovery during phototransduction. The catalytic activity of RetGCs is regulated by their binding to regulatory proteins, guanylate cyclase activating proteins (GCAP1-5) and the retinal degeneration 3 protein (RD3). RetGC1 is activated by its binding to Ca²⁺-free/Mg²⁺-bound GCAP1 at low cytosolic Ca²⁺ levels in light-activated photoreceptors. By contrast, RetGC1 is inactivated by its binding to Ca²⁺-bound GCAP1 and/or RD3 at elevated Ca²⁺ levels in dark-adapted photoreceptors. The Ca²⁺ sensitive cyclase activation helps to replenish the cytosolic cGMP levels in photoreceptors during visual recovery. Mutations in RetGC1, GCAP1 or RD3 that disable the Ca²⁺-dependent regulation of cyclase activity are genetically linked to rod/cone dystrophies and other inherited forms of blindness. Here I review the structural interaction of RetGC1 with GCAP1 and RD3. I propose a two-state concerted model in which the dimeric RetGC1 allosterically switches between active and inactive conformational states with distinct quaternary structures that are oppositely stabilized by the binding of GCAP1 and RD3. The binding of Ca²⁺-free/Mg²⁺-bound GCAP1 is proposed to activate the cyclase by stabilizing RetGC1 in an active conformation (R-state), whereas Ca²⁺-bound GCAP1 and/or RD3 inhibit the cyclase by locking RetGC1 in an inactive conformation (T-state). Exposed hydrophobic residues in GCAP1 (residues H19, Y22, M26, F73, V77, W94) are essential for cyclase activation and could be targeted by rational drug design for the possible treatment of rod/cone dystrophies.

L-type voltage-gated Ca²⁺ channels (CaV1.2 and CaV1.3, called CaV) interact with the Ca²⁺ sensor proteins, calmodulin (CaM) and Ca²⁺ binding Protein 1 (CaBP1), that oppositely control Ca²⁺-dependent channel activity. CaM and CaBP1 can each bind to the IQ-motif within the C-terminal cytosolic domain of CaV, which promotes increased channel open probability under basal conditions. At elevated cytosolic Ca²⁺ levels (caused by CaV channel opening), Ca²⁺-bound CaM binding to CaV is essential for promoting rapid Ca²⁺-dependent channel inactivation (CDI). By contrast, CaV binding to CaBP1 prevents CDI and promotes Ca²⁺-induced channel opening (called CDF). In this review, I provide an overview of the known structures of CaM and CaBP1 and their structural interactions with the IQ-motif to help understand how CaM promotes CDI, whereas CaBP1 prevents CDI and instead promotes CDF. Previous electrophysiology studies suggest that Ca²⁺-free forms of CaM and CaBP1 may pre-associate with CaV under basal conditions. However, previous Ca²⁺ binding data suggest that CaM and CaBP1 are both calculated to bind to Ca²⁺ with an apparent dissociation constant of ~100 nM when CaM or CaBP1 is bound to the IQ-motif. Since the neuronal basal cytosolic Ca²⁺ concentration is ~100 nM, nearly half of the neuronal CaV channels are suggested to be bound to Ca²⁺-bound forms of either CaM or CaBP1 under basal conditions. The pre-association of CaV with calcified forms of CaM or CaBP1 are predicted here to have functional implications. The Ca²⁺-bound form of CaBP1 is proposed to bind to CaV under basal conditions to block CaV binding to CaM, which could explain how CaBP1 might prevent CDI.

The neuronal L-type voltage-gated Ca²⁺ channel (Ca_V1.2) interacts with Ca²⁺ binding protein 1 (CaBP1), that promotes Ca²⁺-induced channel activity. The binding of CaBP1 to the IQ-motif in Ca_V1.2 (residues 1644-1665) blocks the binding of calmodulin and prevents Ca²⁺-dependent inactivation of Ca_V1.2. This Ca²⁺-induced binding of CaBP1 to Ca_V1.2 is important for modulating neuronal synaptic plasticity, which may serve a role in learning and memory. Here we report NMR assignments of the C-terminal domain of CaBP1 (residues 99-167, called CaBP1C) that contains two Ca²⁺ bound at the third and fourth EF-hands (EF3 and EF4) and is bound to the Ca_V1.2 IQ-motif from Ca_V1.2 (BMRB accession no. 51518).

The Ca²⁺ sensor protein, calmodulin (CaM) is ubiquitously expressed in all cells where it binds to hundreds of different target proteins, including dozens of enzymes, receptors, ion channels and numerous Ca²⁺ transporters. The only published NMR chemical shift assignments for Ca²⁺-bound CaM (in the absence of a target) have been determined under acidic conditions: at pH 6.5/310 K (BMRB 6541) and pH 6.3/320 K (BMRB 547). However, some CaM/target complexes are not soluble under these conditions. Also, amide chemical shifts are very sensitive to pH and temperature, which can cause large baseline errors when using the existing chemical shift assignments of free CaM to calculate chemical shift perturbations caused by target binding at neutral pH and physiological temperature. We report complete NMR chemical shift assignments of Ca²⁺-saturated CaM under a set of standard conditions at neutral pH and 308 K that will enable more accurate chemical shift comparison between free CaM and CaM/target complexes (BMRB 51289).

Retinal membrane guanylyl cyclase (RetGC) in photoreceptor rod and cone cells is regulated by a family of guanylyl cyclase activating proteins (GCAP1-7). GCAP5 is expressed in zebrafish photoreceptors and promotes Ca²⁺-dependent regulation of RetGC enzymatic activity that regulates visual phototransduction. We report NMR chemical shift assignments of the Ca²⁺-free activator form of GCAP5 (BMRB No. 27705).

Retinal cyclic nucleotide-gated (CNG) channels consist of two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to two separate sites within the cytosolic region of CNGB1: CaM binding to an N-terminal site (human CNGB1 residues 565-587, called CaM1) decreases the open probability of CNG channels at elevated Ca²⁺ levels in dark-adapted photoreceptors, whereas CaM binding to a separate C-terminal site (CNGB1 residues 1120-1147, called CaM2) may increase channel open probability in light activated photoreceptors. We recently reported NMR chemical shift assignments of Ca²⁺-saturated CaM bound to the CaM1 site of CNGB1 (BMRB no. 51222). Here, we report complete NMR chemical shift assignments of Ca²⁺-saturated CaM bound to the C-terminal CaM2 site of CNGB1 (BMRB no. 51447).

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.

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).