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Structural Characterization of Calmodulin Disease Mutations
108a
Sunday, February 12, 2017
in Cav1.4 are associated with multiple vision disorders including congenital stationary night blindness (CSNB2). Cav1.4 does not undergo Ca2þ-dependent inactivation (CDI) – a negative feedback mechanism seen for other L-type channels mediated by calmodulin (CaM) binding to a consensus IQ-domain in proximal C-terminal domain (pCT) of the pore-forming subunit. The lack of CDI in Cav1.4 is due to a C-terminal automodulatory domain (CTM), located in the distal CT. The CTM is thought to suppress CDI of Cav1.4 channels by competing with CaM-binding to sites in the pCT. A CSNB2-causing mutation (K1591X) in Cav1.4 that deletes the CTM promotes CaM-binding and CDI, but also causes channel activation at more negative potentials than full-length channels (Cav1.4FL). Here, we demonstrate that similar properties are exhibited by a naturally occurring human Cav1.4 splice variant lacking exon 47 (Cav1.4Dex47), and characterized an unexpected role for CaM in the regulation of this channel. By qPCR, we found that Cav1.4Dex47, which lacks the initial 43 amino acids of the CTM, is expressed in primate but not mouse retina. In electrophysiological recordings of transfected HEK293T cells, Cav1.4Dex47 Ca2þ currents activate at more negative voltages and display stronger CDI than Cav1.4FL, similar to K1591X. These effects were blunted by IQdomain mutations known to disrupt CaM-binding to Cav1.4. Mutations that prevent Ca2þ-binding to either N- or C-terminal CaM lobes suppress CDI of Cav1.4Dex47. However, mutations in the N-terminal but not the C-terminal lobe of CaM abolish the effect of exon 47 deletion on channel activation. We conclude that exon 47 contains key molecular determinants within the CTM for regulating CDI and activation, and that CaM plays distinct roles in these processes. 538-Pos Board B303 Structural Characterization of Calmodulin Disease Mutations Kaiqian Wang1, Jocelyn Lu1, Kamilla T. Larsen2, Michael T. Overgaard2, Filip Van Petegem1. 1 University of British Columbia, Vancouver, BC, Canada, 2Aalborg University, Aalborg, Denmark. Calmodulin (CaM) is a ubiquitous calcium-sensing protein involved in the propagation of intracellular calcium signals and the regulation of events ranging from muscle contraction to cell excitability. The human genome contains three CaM genes (CALM 1-3) which encode for protein with identical primary sequences. Despite the redundancy of CaM, single missense mutations in even one of the six alleles are associated with disease phenotypes such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and early-onset severe long QT syndrome (esLQT). CPVT can lead to stress- and exercise-induced arrhythmias and sudden cardiac death; esLQT is characterized by a prolonged QT interval which can also result in ventricular fibrillation. Despite the devastating genetic disorders associated with CaM mutations, the molecular mechanisms by which these mutations manifest into dominant disease phenotypes have yet to be elucidated. Here, we present the crystal structures of several CaM disease mutants, one of which represents a novel CaM conformation not previously characterized. Significant structural changes are observed in both EF-hands III and IV of the C-lobe. In particular, the mutation disrupts the calcium coordination network in EF-hand III and results in abolished calcium binding, leading to CaM that resembles an intermediate between the Ca2þ-CaM and apo-CaM states. In contrast, structures of other disease mutants revealed CaM conformations that closely resemble the wild type structure, with little positional shift in the EF-hand helices of either the N- or C-lobe. These structures can help explain the diverse effects of CaM mutations and the associated disease mechanisms, especially as CaM mutations have differential effects on the function of ryanodine receptor 2 and calcium-dependent inactivation of L-type calcium channels. 539-Pos Board B304 Strontium and Barium in Aqueous Solution and an Ion Channel Blocking Site Mangesh Chaudhari, Susan Rempe. Sandia National Labs, Albuquerque, NM, USA. Ion hydration structure and free energy establish criteria for understanding selective ion binding in potassium (Kþ) ion chan- nels, and may be significant to understanding blocking mechanisms as well. Recently, we investigated the hydration properties of Ba2þ, the most potent blocker of Kþ channels among the simple metal ions. Here, we use a similar method of combining ab initio molecular dynamics simulations, statistical mechanical theory, and electronic structure calculations to probe the fun- damental hydration properties of Sr2þ, which does not block bacterial Kþ channels. The radial distribution of
water around Sr2þ suggests a stable 8-fold geometry in the local hydration environment, similar to Ba2þ. While the predicted hydration free energy of 331.8 kcal/mol is comparable with the experimental result of 334 kcal/mol, the value is significantly more favorable than the 305 kcal/mol hydration free energy of Ba2þ. When placed in the innermost Kþ channel blocking site, the solvation free energies and lowest energy structures of both Sr2þ and Ba2þ are nearly unchanged compared with their respective hydration properties. That result suggests that differences in blocking behavior may arise due to kinetic properties associated with exchange of water ligands for channel ligands instead of equilibrium thermodynamic properties. 540-Pos Board B305 The Calcium Channel A2D Subunit Increases the Gating Charges of Cav1.2 Channels Gustavo F. Contreras1, Nicoletta Savalli2, Antonios Pantazis2, Carlos Gonzalez1, Riccardo Olcese2, Alan Neely1. 1 Centro de Neurociencias de Valparaiso, Valparaiso, Chile, 2Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine at UCLA, LA, CA, USA. Optically tracking the movement of individual voltage sensors (VSD) of human CaV1.2 revealed that association of a2d-1 with a1C resulted in a substantial change in the intrinsic voltage-sensing properties of VSDs I-III (Savalli et al 2016, JGP 142, 147-159). Co-expression of a2d-1 increased the sensitivity of activation for several VSDs indicating that this mostly extracellular subunit increased the electrical distances traversed by voltage-sensing charges. In this study we measured the slope of the voltage-dependence of the channel’s probability of being open (Po) at extremely low probability using marcropatches of Xenopus oocytes expressing CaV1.2/CaVb3 by themselves or in combination with a2d-1. Patches containing from 50 to several 100 channels were recorded in 75 mM Ba2þ and in the presence of 1 mM (-) Bay K 8644 and held at several voltages ranging from 70 to 20 mV for 10 to 90 s. Po times the number of channels (NPo) were obtained by finding the most likely combination of normal distributions shifted by the single channel currents amplitude that described the data. The relative weight of the different distributions was then contrasted with what should be expected from a Poisson distribution of opening levels. The number of channels (N) was estimated from the noise analysis of hundreds of tail current traces at 40 mV following a depolarizing pulse to þ80 mV. In the presence of a2d-1, the limiting slope was about 4 elementary charges, close to the sum of the voltage-dependencies of VSDII and VSDIII activation and clearly less than the sum of all VSDs and thus consistent with the idea that only a subset of VSDs contribute to the effective charges for channel opening. When the a2d-1 subunit was absent the limiting slope was reduced by about one elementary charge. This modest change can be accounted by a reduction in the electrical distance that gating charges need to cross for channel opening. Funding: FONDECYT 3140590 (GC) and 1120864 (AN), R01GM110276 (RO) and 16POST27250284 (NS). 541-Pos Board B306 Calmodulin and Stac3 Enhance Functional Expression of CaV1.1 Jacqueline Niu, Manu Ben Johny, David T. Yue, Takanari Inoue. Johns Hopkins University, Baltimore, MD, USA. CaV1.1 is a prominent L-type voltage-gated calcium channel (VGCC) that plays an integral role in mediating skeletal muscle excitation-contraction coupling. Unlike other homologous L-type channels (e.g. CaV1.3), in depth biophysical analysis of CaV1.1 is challenging as functional expression of these channels has been generally restricted to cell types with a muscular lineage. Interestingly, in contrast to CaV1.3, the carboxy terminus of CaV1.1 has a low affinity for the calcium binding protein, calmodulin (CaM) suggesting that the loss of CaM pre-association may be responsible for the poor functional expression of CaV1.1 in recombinant systems. Here, we explicitly demonstrate that restoration of CaM to the channel complex enables functional expression of CaV1.1 in HEK293 cells. Further mechanistic analysis shows that CaM substantially enhances surface membrane trafficking of CaV1.1. In conjunction with recent studies that showed Stac3 (SH3 and cysteine rich domain 3) adaptor proteins also enable CaV1.1 currents in recombinant systems (Polster et al (2015) PNAS 112:602), our results argue that multiple cell signaling molecules can evoke similar functional outcomes and points to redundancy in molecular pathways that orchestrate CaV1.1 surface expression. These results also suggest there may be an overlap between channel regulation and trafficking of L-type VGCC with Stac3 and CaM. In all, our findings furnish a convenient platform to probe CaV1.1 function and related pharmacology.
Sunday, February 12, 2017
in Cav1.4 are associated with multiple vision disorders including congenital stationary night blindness (CSNB2). Cav1.4 does not undergo Ca2þ-dependent inactivation (CDI) – a negative feedback mechanism seen for other L-type channels mediated by calmodulin (CaM) binding to a consensus IQ-domain in proximal C-terminal domain (pCT) of the pore-forming subunit. The lack of CDI in Cav1.4 is due to a C-terminal automodulatory domain (CTM), located in the distal CT. The CTM is thought to suppress CDI of Cav1.4 channels by competing with CaM-binding to sites in the pCT. A CSNB2-causing mutation (K1591X) in Cav1.4 that deletes the CTM promotes CaM-binding and CDI, but also causes channel activation at more negative potentials than full-length channels (Cav1.4FL). Here, we demonstrate that similar properties are exhibited by a naturally occurring human Cav1.4 splice variant lacking exon 47 (Cav1.4Dex47), and characterized an unexpected role for CaM in the regulation of this channel. By qPCR, we found that Cav1.4Dex47, which lacks the initial 43 amino acids of the CTM, is expressed in primate but not mouse retina. In electrophysiological recordings of transfected HEK293T cells, Cav1.4Dex47 Ca2þ currents activate at more negative voltages and display stronger CDI than Cav1.4FL, similar to K1591X. These effects were blunted by IQdomain mutations known to disrupt CaM-binding to Cav1.4. Mutations that prevent Ca2þ-binding to either N- or C-terminal CaM lobes suppress CDI of Cav1.4Dex47. However, mutations in the N-terminal but not the C-terminal lobe of CaM abolish the effect of exon 47 deletion on channel activation. We conclude that exon 47 contains key molecular determinants within the CTM for regulating CDI and activation, and that CaM plays distinct roles in these processes. 538-Pos Board B303 Structural Characterization of Calmodulin Disease Mutations Kaiqian Wang1, Jocelyn Lu1, Kamilla T. Larsen2, Michael T. Overgaard2, Filip Van Petegem1. 1 University of British Columbia, Vancouver, BC, Canada, 2Aalborg University, Aalborg, Denmark. Calmodulin (CaM) is a ubiquitous calcium-sensing protein involved in the propagation of intracellular calcium signals and the regulation of events ranging from muscle contraction to cell excitability. The human genome contains three CaM genes (CALM 1-3) which encode for protein with identical primary sequences. Despite the redundancy of CaM, single missense mutations in even one of the six alleles are associated with disease phenotypes such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and early-onset severe long QT syndrome (esLQT). CPVT can lead to stress- and exercise-induced arrhythmias and sudden cardiac death; esLQT is characterized by a prolonged QT interval which can also result in ventricular fibrillation. Despite the devastating genetic disorders associated with CaM mutations, the molecular mechanisms by which these mutations manifest into dominant disease phenotypes have yet to be elucidated. Here, we present the crystal structures of several CaM disease mutants, one of which represents a novel CaM conformation not previously characterized. Significant structural changes are observed in both EF-hands III and IV of the C-lobe. In particular, the mutation disrupts the calcium coordination network in EF-hand III and results in abolished calcium binding, leading to CaM that resembles an intermediate between the Ca2þ-CaM and apo-CaM states. In contrast, structures of other disease mutants revealed CaM conformations that closely resemble the wild type structure, with little positional shift in the EF-hand helices of either the N- or C-lobe. These structures can help explain the diverse effects of CaM mutations and the associated disease mechanisms, especially as CaM mutations have differential effects on the function of ryanodine receptor 2 and calcium-dependent inactivation of L-type calcium channels. 539-Pos Board B304 Strontium and Barium in Aqueous Solution and an Ion Channel Blocking Site Mangesh Chaudhari, Susan Rempe. Sandia National Labs, Albuquerque, NM, USA. Ion hydration structure and free energy establish criteria for understanding selective ion binding in potassium (Kþ) ion chan- nels, and may be significant to understanding blocking mechanisms as well. Recently, we investigated the hydration properties of Ba2þ, the most potent blocker of Kþ channels among the simple metal ions. Here, we use a similar method of combining ab initio molecular dynamics simulations, statistical mechanical theory, and electronic structure calculations to probe the fun- damental hydration properties of Sr2þ, which does not block bacterial Kþ channels. The radial distribution of
water around Sr2þ suggests a stable 8-fold geometry in the local hydration environment, similar to Ba2þ. While the predicted hydration free energy of 331.8 kcal/mol is comparable with the experimental result of 334 kcal/mol, the value is significantly more favorable than the 305 kcal/mol hydration free energy of Ba2þ. When placed in the innermost Kþ channel blocking site, the solvation free energies and lowest energy structures of both Sr2þ and Ba2þ are nearly unchanged compared with their respective hydration properties. That result suggests that differences in blocking behavior may arise due to kinetic properties associated with exchange of water ligands for channel ligands instead of equilibrium thermodynamic properties. 540-Pos Board B305 The Calcium Channel A2D Subunit Increases the Gating Charges of Cav1.2 Channels Gustavo F. Contreras1, Nicoletta Savalli2, Antonios Pantazis2, Carlos Gonzalez1, Riccardo Olcese2, Alan Neely1. 1 Centro de Neurociencias de Valparaiso, Valparaiso, Chile, 2Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine at UCLA, LA, CA, USA. Optically tracking the movement of individual voltage sensors (VSD) of human CaV1.2 revealed that association of a2d-1 with a1C resulted in a substantial change in the intrinsic voltage-sensing properties of VSDs I-III (Savalli et al 2016, JGP 142, 147-159). Co-expression of a2d-1 increased the sensitivity of activation for several VSDs indicating that this mostly extracellular subunit increased the electrical distances traversed by voltage-sensing charges. In this study we measured the slope of the voltage-dependence of the channel’s probability of being open (Po) at extremely low probability using marcropatches of Xenopus oocytes expressing CaV1.2/CaVb3 by themselves or in combination with a2d-1. Patches containing from 50 to several 100 channels were recorded in 75 mM Ba2þ and in the presence of 1 mM (-) Bay K 8644 and held at several voltages ranging from 70 to 20 mV for 10 to 90 s. Po times the number of channels (NPo) were obtained by finding the most likely combination of normal distributions shifted by the single channel currents amplitude that described the data. The relative weight of the different distributions was then contrasted with what should be expected from a Poisson distribution of opening levels. The number of channels (N) was estimated from the noise analysis of hundreds of tail current traces at 40 mV following a depolarizing pulse to þ80 mV. In the presence of a2d-1, the limiting slope was about 4 elementary charges, close to the sum of the voltage-dependencies of VSDII and VSDIII activation and clearly less than the sum of all VSDs and thus consistent with the idea that only a subset of VSDs contribute to the effective charges for channel opening. When the a2d-1 subunit was absent the limiting slope was reduced by about one elementary charge. This modest change can be accounted by a reduction in the electrical distance that gating charges need to cross for channel opening. Funding: FONDECYT 3140590 (GC) and 1120864 (AN), R01GM110276 (RO) and 16POST27250284 (NS). 541-Pos Board B306 Calmodulin and Stac3 Enhance Functional Expression of CaV1.1 Jacqueline Niu, Manu Ben Johny, David T. Yue, Takanari Inoue. Johns Hopkins University, Baltimore, MD, USA. CaV1.1 is a prominent L-type voltage-gated calcium channel (VGCC) that plays an integral role in mediating skeletal muscle excitation-contraction coupling. Unlike other homologous L-type channels (e.g. CaV1.3), in depth biophysical analysis of CaV1.1 is challenging as functional expression of these channels has been generally restricted to cell types with a muscular lineage. Interestingly, in contrast to CaV1.3, the carboxy terminus of CaV1.1 has a low affinity for the calcium binding protein, calmodulin (CaM) suggesting that the loss of CaM pre-association may be responsible for the poor functional expression of CaV1.1 in recombinant systems. Here, we explicitly demonstrate that restoration of CaM to the channel complex enables functional expression of CaV1.1 in HEK293 cells. Further mechanistic analysis shows that CaM substantially enhances surface membrane trafficking of CaV1.1. In conjunction with recent studies that showed Stac3 (SH3 and cysteine rich domain 3) adaptor proteins also enable CaV1.1 currents in recombinant systems (Polster et al (2015) PNAS 112:602), our results argue that multiple cell signaling molecules can evoke similar functional outcomes and points to redundancy in molecular pathways that orchestrate CaV1.1 surface expression. These results also suggest there may be an overlap between channel regulation and trafficking of L-type VGCC with Stac3 and CaM. In all, our findings furnish a convenient platform to probe CaV1.1 function and related pharmacology.