scholarly journals Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels

2013 ◽  
Vol 142 (4) ◽  
pp. 381-404 ◽  
Author(s):  
Kerstin Vocke ◽  
Kristin Dauner ◽  
Anne Hahn ◽  
Anne Ulbrich ◽  
Jana Broecker ◽  
...  
Keyword(s):  
Splice Variant ◽  
Chloride Channels ◽  
Cell Types ◽  
Binding Motif ◽  
Binding Studies ◽  
Channel Activation ◽  
Calcium Dependent ◽  
Calmodulin Binding ◽  
Dependent Inactivation ◽  
Cl Channels

Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca2+/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca2+/calmodulin, one at submicromolar Ca2+ concentrations and one in the micromolar Ca2+ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca2+/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca2+ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca2+ regulation in anoctamin Cl− channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types.

scholarly journals Resolving the molecular fingerprint of the distal carboxy tail in modulating CaV1 calcium dependent inactivation

2021 ◽  
Author(s):  
Lingjie Sang ◽  
Daiana C. O. Vieira ◽  
David T. Yue ◽  
Manu Ben-Johny ◽  
Ivy E. Dick
Keyword(s):  
Cell Types ◽  
Regulatory Process ◽  
Open Probability ◽  
Alanine Scanning Mutagenesis ◽  
Molecular Fingerprint ◽  
Calcium Dependent ◽  
Dependent Inactivation ◽  
Competitive Mechanism ◽  
Molecular Rheostat ◽  
Different Cell Types

AbstractCa2+/calmodulin-dependent inactivation (CDI) of CaV channels is a critical regulatory process required for tuning the kinetics of Ca2+ entry for different cell types and physiologic responses. Calmodulin (CaM) resides on the IQ domain of the CaV carboxy-tail, such that Ca2+ binding initiates a reduction in channel open probability, manifesting as CDI. This regulatory process exerts a significant impact on Ca2+ entry and is tailored by alternative splicing. CaV1.3 and CaV1.4 feature a long-carboxy-tail splice variant that modulates CDI through a competitive mechanism. In these channels, the distal-carboxy-tail (DCT) harbors an inhibitor of CDI (ICDI) module that competitively displaces CaM from the IQ domain, thereby diminishing CDI. While this overall mechanism is now well-described, the detailed interaction loci for ICDI binding to the IQ domain is yet to be elucidated. Here, we perform alanine-scanning mutagenesis of the IQ and ICDI domains and evaluate the contribution of neighboring regions. We identify multiple critical residues within the IQ domain, ICDI and the nearby A region of the channel, which are required for high affinity IQ/ICDI binding. Importantly, disruption of this interaction commensurately diminishes ICDI function, as seen by the re-emergence of CDI in mutant channels. Furthermore, analysis of the homologous ICDI region of CaV1.2 reveals a selective effect of this channel region on CaV1.3 channels, implicating a cross-channel modulatory scheme in cells expressing both channel subtypes. In all, these findings provide new insights into a molecular rheostat that fine tunes Ca2+ entry and supports normal neuronal and cardiac function.

Calcium-dependent regulation of airway epithelial chloride channels

1990 ◽  
Vol 258 (2) ◽  
pp. L25-L32 ◽  
Author(s):  
J. P. Clancy ◽  
J. D. McCann ◽  
M. Li ◽  
M. J. Welsh
Keyword(s):  
Chloride Channels ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Intact Cells ◽  
Channel Activation ◽  
Tracheal Epithelial Cells ◽  
Cl Channel ◽  
Cl Channels ◽  
Free Membrane

To determine how cell calcium ([Ca2+]c) regulates apical Cl- channels, we measured the rate of 125-Iodide (125I-) efflux to assay Cl- channel activity in intact cells and examined cell-free membrane patches from cultured canine tracheal epithelial cells. The Ca2+ elevating agonist bradykinin and the calcium ionophore A23187 increased 125I- efflux. This response did not require prostaglandin production. Under several conditions, changes in [Ca2+]c were temporally dissociated from changes in channel activation: a transient increase in [Ca2+]c caused a prolonged stimulation of 125I- efflux. Neither Cl- channel activation nor open-channel probability was affected by varying internal [Ca2+] in excised membrane patches. Adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca2(+)-dependent channel activation may be independent: cAMP-stimulated 125I- efflux did not require an increase in [Ca2+]c, Ca2(+)-stimulated efflux did not require an increase in cAMP, and simultaneous addition of A23187 and isoproterenol produced additive effects on 125I- efflux. The data suggest that an increase in [Ca2+]c activates Cl- channels, however, the effect of Ca2+ appears to be indirect, not involving a ligand-type interaction with the channel.

Small linear chloride channels are endogenous to nonepithelial cells

1992 ◽  
Vol 263 (3) ◽  
pp. C708-C713 ◽  
Author(s):  
S. E. Gabriel ◽  
E. M. Price ◽  
R. C. Boucher ◽  
M. J. Stutts
Keyword(s):  
Cell Lines ◽  
Chloride Channels ◽  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Ovary Cells ◽  
3T3 Fibroblasts ◽  
Whole Cell Patch Clamp ◽  
Cl Channels

We used both single-channel and whole cell patch-clamp techniques to characterize chloride channels and currents endogenous to Sf9 cells, 3T3 fibroblasts, and Chinese hamster ovary cells. In cell-attached patches from these cell types, anion channels were observed with low ohmic conductance (4-11 ps), linear current-voltage relationships, and little time- or voltage-dependent behavior. These channels are very similar to the Cl- channels reported to appear concomitant with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in these cell lines. The presence of such endogenous channels suggests either that low levels of CFTR are present in all of these cell lines prior to transfection or that an endogenous non-CFTR channel is present in these cell types. Our results suggest that at least some of the channel behaviors attributed to expressed, recombinant CFTR in previous studies may have been due to these endogenous Cl- channels.

scholarly journals Sequence Differences in the IQ Motifs of CaV1.1 and CaV1.2 Strongly Impact Calmodulin Binding and Calcium-dependent Inactivation

2008 ◽  
Vol 283 (43) ◽  
pp. 29301-29311 ◽  
Author(s):  
Joshua Ohrtman ◽  
Barbara Ritter ◽  
Alexander Polster ◽  
Kurt G. Beam ◽  
Symeon Papadopoulos
Keyword(s):  
Calcium Dependent ◽  
Calmodulin Binding ◽  
Dependent Inactivation

scholarly journals Intracellular regulators of neuronal sprouting: calmodulin-binding proteins of nerve growth cones.

1985 ◽  
Vol 101 (3) ◽  
pp. 1153-1160 ◽  
Author(s):  
C Hyman ◽  
K H Pfenninger
Keyword(s):  
Growth Cone ◽  
Binding Proteins ◽  
Growth Cones ◽  
Binding Studies ◽  
Nerve Growth ◽  
Calcium Dependent ◽  
Calmodulin Binding ◽  
Nerve Growth Cone ◽  
The Individual ◽  
Neuronal Sprouting

The focus of this study is a quantitative biochemical analysis of the calcium-dependent interactions of calmodulin with a nerve growth cone preparation from fetal rat brain (Pfenninger, K. H., L. Ellis, M. P. Johnson, L. B. Freidman, and S. Somlo, 1983, Cell 35:573-584). The presence of calmodulin as an integral component of this preparation is demonstrated, and quantitative binding studies are presented. The binding of 125I-calmodulin to nerve growth cone material is shown to be highly specific, calcium dependent, and saturable at nanomolar calmodulin concentrations. Additionally, the growth cones' binding components appear to be membrane proteins. The individual molecular mass species of growth cone proteins displaying calcium-dependent calmodulin binding are also detailed and presented in comparison with those of synaptosomes. This analysis reveals differences between the calmodulin binding proteins of the growth cone preparation and the synaptosome fraction, suggesting the presence in growth cones of a specialized set of components which may be involved in regulatory mechanisms controlling neuritic sprouting.

scholarly journals A CACNA1A variant associated with trigeminal neuralgia alters the gating of Cav2.1 channels

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Eder Gambeta ◽  
Maria A. Gandini ◽  
Ivana A. Souza ◽  
Laurent Ferron ◽  
Gerald W. Zamponi
Keyword(s):  
Trigeminal Neuralgia ◽  
Missense Mutation ◽  
Neurotransmitter Release ◽  
Trigeminal System ◽  
Wild Type ◽  
Calcium Dependent ◽  
Whole Cell Patch Clamp ◽  
C Terminus ◽  
Dependent Inactivation

AbstractA novel missense mutation in the CACNA1A gene that encodes the pore forming α1 subunit of the CaV2.1 voltage-gated calcium channel was identified in a patient with trigeminal neuralgia. This mutation leads to a substitution of proline 2455 by histidine (P2455H) in the distal C-terminus region of the channel. Due to the well characterized role of this channel in neurotransmitter release, our aim was to characterize the biophysical properties of the P2455H variant in heterologously expressed CaV2.1 channels. Whole-cell patch clamp recordings of wild type and mutant CaV2.1 channels expressed in tsA-201 cells reveal that the mutation mediates a depolarizing shift in the voltage-dependence of activation and inactivation. Moreover, the P2455H mutant strongly reduced calcium-dependent inactivation of the channel that is consistent with an overall gain of function. Hence, the P2455H CaV2.1 missense mutation alters the gating properties of the channel, suggesting that associated changes in CaV2.1-dependent synaptic communication in the trigeminal system may contribute to the development of trigeminal neuralgia.

scholarly journals Binding Studies Reveal Phospholipid Specificity and Its Role in the Calcium-Dependent Mechanism of Action of Daptomycin

2021 ◽  
Author(s):  
Ioli Kotsogianni ◽  
Thomas M. Wood ◽  
Francesca M. Alexander ◽  
Stephen A. Cochrane ◽  
Nathaniel I. Martin
Keyword(s):  
Mechanism Of Action ◽  
Binding Studies ◽  
Calcium Dependent ◽  
Dependent Mechanism

Altered frequency-dependent inactivation and steady-state inactivation of polyglutamine-expanded α1A in SCA6

2007 ◽  
Vol 292 (3) ◽  
pp. C1078-C1086 ◽  
Author(s):  
Haiyan Chen ◽  
Erika S. Piedras-Rentería
Keyword(s):  
Steady State ◽  
Late Onset ◽  
Spinocerebellar Ataxia Type ◽  
Gain Of Function ◽  
Calcium Dependent ◽  
Spinocerebellar Ataxia Type 6 ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Activity Dependent

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease of the cerebellum and inferior olives characterized by a late-onset cerebellar ataxia and selective loss of Purkinje neurons ( 15 , 16 ). SCA6 arises from an expansion of the polyglutamine tract located in exon 47 of the α1A (P/Q-type calcium channel) gene from a nonpathogenic size of 4 to 18 glutamines (CAG4–18) to CAG19–33 in SCA6. The molecular basis of SCA6 is poorly understood. To date, the biophysical properties studied in heterologous systems support both a gain and a loss of channel function in SCA6. We studied the behavior of the human α1A isoform, previously found to elicit a gain of function in disease ( 41 ), focusing on properties in which the COOH terminus of the channel is critical for function: we analyzed the current properties in the presence of β4- and β2a-subunits (both known to interact with the α1A COOH terminus), current kinetics of activation and inactivation, calcium-dependent inactivation and facilitation, voltage-dependent inactivation, frequency dependence, and steady-state activation and inactivation properties. We found that SCA6 channels have decreased activity-dependent inactivation and a depolarizing shift (+6 mV) in steady-state inactivation properties consistent with a gain of function.

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