Pflugers Archiv : European journal of physiology, 2017
Inactivation of L-type calcium channel (Cav1.2) is an important determinant of the length of the ... more Inactivation of L-type calcium channel (Cav1.2) is an important determinant of the length of the cardiac action potential. Here, we report a key role of the voltage-sensing segment IS4 in Cav1.2 inactivation. Neutralization of IS4 charges gradually shifted the steady-state inactivation curve on the voltages axis from 5.1 ± 3.7 mV in single point mutant IS4(K1Q) to -26.7 ± 1.3 mV in quadruple mutant IS4(K1Q/R2Q/R3Q/R4Q) compared to wild-type (WT) and accelerated inactivation. The slope factor of the Boltzmann curve of inactivation was decreased from 17.4 ± 3.5 mV (IS4(K1Q)) to 6.2 ± 0.7 mV (IS4(K1Q/R2Q/R3Q/R4Q)). Neutralizations of single or multiple charges in IIS4 and IIIS4 did not significantly affect the time course of inactivation. Neutralization of individual IVS4 charges shifted the inactivation curve between 17.4 ± 1.7 mV (IVS4(R2Q)) and -4.6 ± 1.4 mV (IVS4(R4Q)) on the voltage axis and affected the slope of the inactivation curves (IVS4(R2Q): 10.2 ± 1.2 mV, IVS4(R4Q): 9.7 ± ...
hERG (Kv11.1) channel inhibition is a widely accepted predictor of cardiac arrhythmia. hERG chann... more hERG (Kv11.1) channel inhibition is a widely accepted predictor of cardiac arrhythmia. hERG channel inhibition alone is often insufficient to predict pro-arrhythmic drug effects. This study used a library of dofetilide derivatives to investigate the relationship between standard measures of hERG current block in an expression system and changes in action potential duration (APD) in hiPSC-CMs. The interference from accompanying block of Cav1.2 and Nav1.5 channels was investigated along with an in-silico action potential (AP) model. Drug-induced changes in APD were assessed in hiPSC-CMs using voltage sensitive dyes. The half-maximal inhibitory concentrations (IC50 ) of hERG current by dofetilide and 13 derivatives were estimated in a HEK293 expression system. The relative potency of the drug on APD was estimated by calculating the drug dose (D150 ) required to prolong the APD at 90% (APD90 ) repolarisation by 50%. The D150 in hiPSC-CMs was linearly correlated with IC50 of hERG current...
Pflugers Archiv : European journal of physiology, 2016
In order to specify the role of individual S4 segments in CaV1.2 gating, charged residues of segm... more In order to specify the role of individual S4 segments in CaV1.2 gating, charged residues of segments IS4-IVS4 were replaced by glutamine and the corresponding effects on activation/deactivation of calcium channel currents were analysed. Almost all replacements of charges in IS4 and IIIS4 decreased the slope of the Boltzmann curve of channel activation (activation curve) while charge neutralisations in IIS4 and IVS4 did not significantly affect the slope. S4 mutations caused either left or rightward shifts of the activation curve, and in wild-type channels, these S4 mutations hardly affected current kinetics.In slowly gating pore (S6) mutants (G432W, A780T, G1193T or A1503G), neutralisations in S4 segments significantly accelerated current kinetics. Likewise in wild type, charge replacements in IS4 and IIIS4 of pore mutants reduced the slope of the activation curves while substitutions of charges in IIS4 and IVS4 had less or no impact. We propose a gating model where the structurall...
hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes.... more hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes. hERG R56Q channels were expressed in hiPSCderived cardiomyocytes (hiPSC-CMs) and electrical properties of the cells were measured with whole-cell patch-clamp. We found that, like in nonmyocyte cells, hERG R56Q had fast deactivation kinetics when expressed in hiPSC-CMs. We report here that i-eag domains slowed the deactivation kinetics in hERG R56Q currents in cardiomyocytes by making a direct association with hERG R56Q channels, as measured with FRET spectroscopy, which means that the modulatory role of i-eag domains function in the environment of cardiomyocytes. Isolated eag domains rescued the dysfunction in hERG R56Q channels in the cellular environment of a cardiomyocyte, which shows that they may be useful as a biological therapeutic.
Page 203. Chapter 8 Estimation of Drug Affinities for Calcium Channel Conformational States Steff... more Page 203. Chapter 8 Estimation of Drug Affinities for Calcium Channel Conformational States Steffen Hering'and Eugen Nikolaevich Timin1 'lnstitute for Biochemical Pharmacology lnnsbruck, Austria fA. ... 1973; Courtney, 1975; Khodorov et al. ...
Journal of Chemical Information and Modeling, 2014
electrode voltage clamp. ABSTRACT. K + channels play a critical role in numerous physiological an... more electrode voltage clamp. ABSTRACT. K + channels play a critical role in numerous physiological and pathophysiological processes rendering them an attractive target for therapeutic intervention. However, the hERG K + channel poses a special challenge in drug discovery, since block of this channel by a plethora of diverse chemical entities can lead to long QT syndrome and sudden death. Of particular interest is the so called trapping phenomenon, characterized by capture of a drug behind closed channel gates, which harbors an increased pro-arrhythmic risk. In this study we investigated the influence of trapped blockers on the gating dynamics and probed the state dependence of dissociation in K + channels by making use of the quaternary tetrabutylammonium. By applying essential dynamics simulations and two-electrode voltage clamp we obtained detailed insights into the dynamics of trapping in KcsA and hERG. Our simulations suggest that the trapped TBA influences the F656 flexibility during gate closure. Based on these findings, we provide a
Proceedings of the National Academy of Sciences, 1997
The role of channel inactivation in the molecular mechanism of calcium (Ca 2+ ) channel block by ... more The role of channel inactivation in the molecular mechanism of calcium (Ca 2+ ) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca 2+ channels that carry the high affinity determinants of the PAA receptor site [Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995) J. Biol. Chem. 270, 22119–22122] but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca 2+ channels in Xenopus oocytes. Substitution of single putative pore-orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (I Ba ) by (−)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (−)D600 slowed I Ba recovery after maintain...
Pflügers Archiv - European Journal of Physiology, 2012
Voltage sensors trigger the closed-open transitions in the pore of voltage-gated ion channels. To... more Voltage sensors trigger the closed-open transitions in the pore of voltage-gated ion channels. To probe the transmission of voltage sensor signalling to the channel pore of Ca V 1.2, we investigated how elimination of positive charges in the S4 segments (charged residues were replaced by neutral glutamine) modulates gating perturbations induced by mutations in pore-lining S6 segments. Neutralisation of all positively charged residues in IIS4 produced a functional channel (IIS4 N), while replacement of the charged residues in IS4, IIIS4 and IVS4 segments resulted in nonfunctional channels. The IIS4 N channel displayed activation kinetics similar to wild type. Mutations in a highly conserved structure motif on S6 segments ("GAGA ring": G432W in IS6, A780T in IIS6, G1193T in IIIS6 and A1503G in IVS6) induce strong left-shifted activation curves and decelerated channel deactivation kinetics. When IIS4 N was combined with these mutations, the activation curves were shifted back towards wild type and current kinetics were accelerated. In contrast, 12 other mutations adjacent to the GAGA ring in IS6-IVS6, which also affect activation gating, were not rescued by IIS4 N. Thus, the rescue of gating distortions in segments IS6-IVS6 by IIS4 N is highly position-specific. Thermodynamic cycle analysis supports the hypothesis that IIS4 is energetically coupled with the distantly located GAGA residues. We speculate that conformational changes caused by neutralisation of IIS4 are not restricted to domain II (IIS6) but are transmitted to gating structures in domains I, III and IV via the GAGA ring.
Point mutations in pore-lining S6 segments of CaV1.2 shift the voltage dependence of activation i... more Point mutations in pore-lining S6 segments of CaV1.2 shift the voltage dependence of activation into the hyperpolarizing direction and significantly decelerate current activation and deactivation. Here, we analyze theses changes in channel gating in terms of a circular four-state model accounting for an activation R–A–O and a deactivation O–D–R pathway. Transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements. Voltage-independent pore openings and closures during activation (A–O) and deactivation (D–R) are described by rate constants α and β, and γ and δ, respectively. Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)). Estimated model parameters of 15 CaV1.2 constructs well describe the activation and dea...
Voltage dependence and kinetics of Ca V 1.2 activation are affected by structural changes in pore... more Voltage dependence and kinetics of Ca V 1.2 activation are affected by structural changes in pore-lining S6 segments of the ␣ 1-subunit. Significant effects are induced by either proline or threonine substitutions in the lower third of segment IIS6 ("bundle crossing region"), where S6 segments are likely to seal the channel in the closed conformation (Hohaus, A.
4 The abbreviations used are: GFP, green fluorescent protein; WT, wild-type; NaChBac, bacterial s... more 4 The abbreviations used are: GFP, green fluorescent protein; WT, wild-type; NaChBac, bacterial sodium channel; Bay K8644, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)pyridine-3-carboxylic acid methyl ester.
The inner cavity of the hERG potassium ion channel can accommodate large, structurally diverse co... more The inner cavity of the hERG potassium ion channel can accommodate large, structurally diverse compounds that can be trapped in the channel by closure of the activation gate. A small set of propafenone derivatives was synthesized, and both use-dependency and recovery from block were tested in order to gain insight into the behavior of these compounds with respect to trapping and non-trapping. Ligand-protein docking into homology models of the closed and open state of the hERG channel provides the first evidence for the molecular basis of drug trapping.
A channelopathy mutation in segment IIS6 of Ca V 1.4 (I745T) has been shown to cause severe visua... more A channelopathy mutation in segment IIS6 of Ca V 1.4 (I745T) has been shown to cause severe visual impairment by shifting the activation and inactivation curves to more hyperpolarized voltages and slowing activation and inactivation kinetics. A similar gating phenotype is caused by the corresponding mutation, I781T, in Ca V 1.2 (midpoint of activation curve (V 0.5) shifted to-37.7 ± 1.2 mV). We show here that wild-type gating can partially be restored by a helix stabilizing rescue mutation N785A. V 0.5 of I781T/N785A (V 0.5 =-21.5 ± 0.6 mV) was shifted back towards wild-type (V 0.5 =-9.9 ± 1.1 mV). Homology models developed in our group (see accompanying article for details) were used to perform Molecular Dynamics-simulations (MD-simulations) on wild-type and mutant channels. Systematic changes in segment IIIS6 (M1187-F1194) and in helix IIS6 (N785-L786) were studied. The simulated structural changes in S6 segments of I781T/N785A were less pronounced than in I781T. A delicate balance between helix flexibility and stability enabling the formation of hydrophobic seals at the inner channel mouth appears to be important for wild-type Ca V 1.2 gating. Our study illustrates that effects of mutations in the lower part of IIS6 may not be localized to the residue or even segment being mutated, but may affect conformations of interacting segments.
Ca 2þ /calmodulin-dependent inactivation (CDI) of Ca V channels plays a crucial role in the homeo... more Ca 2þ /calmodulin-dependent inactivation (CDI) of Ca V channels plays a crucial role in the homeostasis of intracellular Ca 2þ , and serves as an ideal prototype for investigating Ca 2þ feedback regulation within biological systems. The prevailing view of CDI mechanism is that Ca 2þ-free calmodulin (apoCaM) preassociates with the IQ domain of the proximal carboxy terminus of channels, and this 'resident' calmodulin (CaM) acts as Ca 2þ sensor to somehow trigger CDI upon Ca 2þ binding. The downstream CaM/channel configurations leading to CDI are complex and under investigation at present. Contrasting with this complexity, Ca V 1.3 and Ca V 1.4 feature a long-carboxy-tail splice variant that minimizes CDI by a beautifully simple mechanism. We recently combined
Pflugers Archiv : European journal of physiology, 2017
Inactivation of L-type calcium channel (Cav1.2) is an important determinant of the length of the ... more Inactivation of L-type calcium channel (Cav1.2) is an important determinant of the length of the cardiac action potential. Here, we report a key role of the voltage-sensing segment IS4 in Cav1.2 inactivation. Neutralization of IS4 charges gradually shifted the steady-state inactivation curve on the voltages axis from 5.1 ± 3.7 mV in single point mutant IS4(K1Q) to -26.7 ± 1.3 mV in quadruple mutant IS4(K1Q/R2Q/R3Q/R4Q) compared to wild-type (WT) and accelerated inactivation. The slope factor of the Boltzmann curve of inactivation was decreased from 17.4 ± 3.5 mV (IS4(K1Q)) to 6.2 ± 0.7 mV (IS4(K1Q/R2Q/R3Q/R4Q)). Neutralizations of single or multiple charges in IIS4 and IIIS4 did not significantly affect the time course of inactivation. Neutralization of individual IVS4 charges shifted the inactivation curve between 17.4 ± 1.7 mV (IVS4(R2Q)) and -4.6 ± 1.4 mV (IVS4(R4Q)) on the voltage axis and affected the slope of the inactivation curves (IVS4(R2Q): 10.2 ± 1.2 mV, IVS4(R4Q): 9.7 ± ...
hERG (Kv11.1) channel inhibition is a widely accepted predictor of cardiac arrhythmia. hERG chann... more hERG (Kv11.1) channel inhibition is a widely accepted predictor of cardiac arrhythmia. hERG channel inhibition alone is often insufficient to predict pro-arrhythmic drug effects. This study used a library of dofetilide derivatives to investigate the relationship between standard measures of hERG current block in an expression system and changes in action potential duration (APD) in hiPSC-CMs. The interference from accompanying block of Cav1.2 and Nav1.5 channels was investigated along with an in-silico action potential (AP) model. Drug-induced changes in APD were assessed in hiPSC-CMs using voltage sensitive dyes. The half-maximal inhibitory concentrations (IC50 ) of hERG current by dofetilide and 13 derivatives were estimated in a HEK293 expression system. The relative potency of the drug on APD was estimated by calculating the drug dose (D150 ) required to prolong the APD at 90% (APD90 ) repolarisation by 50%. The D150 in hiPSC-CMs was linearly correlated with IC50 of hERG current...
Pflugers Archiv : European journal of physiology, 2016
In order to specify the role of individual S4 segments in CaV1.2 gating, charged residues of segm... more In order to specify the role of individual S4 segments in CaV1.2 gating, charged residues of segments IS4-IVS4 were replaced by glutamine and the corresponding effects on activation/deactivation of calcium channel currents were analysed. Almost all replacements of charges in IS4 and IIIS4 decreased the slope of the Boltzmann curve of channel activation (activation curve) while charge neutralisations in IIS4 and IVS4 did not significantly affect the slope. S4 mutations caused either left or rightward shifts of the activation curve, and in wild-type channels, these S4 mutations hardly affected current kinetics.In slowly gating pore (S6) mutants (G432W, A780T, G1193T or A1503G), neutralisations in S4 segments significantly accelerated current kinetics. Likewise in wild type, charge replacements in IS4 and IIIS4 of pore mutants reduced the slope of the activation curves while substitutions of charges in IIS4 and IVS4 had less or no impact. We propose a gating model where the structurall...
hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes.... more hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes. hERG R56Q channels were expressed in hiPSCderived cardiomyocytes (hiPSC-CMs) and electrical properties of the cells were measured with whole-cell patch-clamp. We found that, like in nonmyocyte cells, hERG R56Q had fast deactivation kinetics when expressed in hiPSC-CMs. We report here that i-eag domains slowed the deactivation kinetics in hERG R56Q currents in cardiomyocytes by making a direct association with hERG R56Q channels, as measured with FRET spectroscopy, which means that the modulatory role of i-eag domains function in the environment of cardiomyocytes. Isolated eag domains rescued the dysfunction in hERG R56Q channels in the cellular environment of a cardiomyocyte, which shows that they may be useful as a biological therapeutic.
Page 203. Chapter 8 Estimation of Drug Affinities for Calcium Channel Conformational States Steff... more Page 203. Chapter 8 Estimation of Drug Affinities for Calcium Channel Conformational States Steffen Hering'and Eugen Nikolaevich Timin1 'lnstitute for Biochemical Pharmacology lnnsbruck, Austria fA. ... 1973; Courtney, 1975; Khodorov et al. ...
Journal of Chemical Information and Modeling, 2014
electrode voltage clamp. ABSTRACT. K + channels play a critical role in numerous physiological an... more electrode voltage clamp. ABSTRACT. K + channels play a critical role in numerous physiological and pathophysiological processes rendering them an attractive target for therapeutic intervention. However, the hERG K + channel poses a special challenge in drug discovery, since block of this channel by a plethora of diverse chemical entities can lead to long QT syndrome and sudden death. Of particular interest is the so called trapping phenomenon, characterized by capture of a drug behind closed channel gates, which harbors an increased pro-arrhythmic risk. In this study we investigated the influence of trapped blockers on the gating dynamics and probed the state dependence of dissociation in K + channels by making use of the quaternary tetrabutylammonium. By applying essential dynamics simulations and two-electrode voltage clamp we obtained detailed insights into the dynamics of trapping in KcsA and hERG. Our simulations suggest that the trapped TBA influences the F656 flexibility during gate closure. Based on these findings, we provide a
Proceedings of the National Academy of Sciences, 1997
The role of channel inactivation in the molecular mechanism of calcium (Ca 2+ ) channel block by ... more The role of channel inactivation in the molecular mechanism of calcium (Ca 2+ ) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca 2+ channels that carry the high affinity determinants of the PAA receptor site [Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995) J. Biol. Chem. 270, 22119–22122] but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca 2+ channels in Xenopus oocytes. Substitution of single putative pore-orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (I Ba ) by (−)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (−)D600 slowed I Ba recovery after maintain...
Pflügers Archiv - European Journal of Physiology, 2012
Voltage sensors trigger the closed-open transitions in the pore of voltage-gated ion channels. To... more Voltage sensors trigger the closed-open transitions in the pore of voltage-gated ion channels. To probe the transmission of voltage sensor signalling to the channel pore of Ca V 1.2, we investigated how elimination of positive charges in the S4 segments (charged residues were replaced by neutral glutamine) modulates gating perturbations induced by mutations in pore-lining S6 segments. Neutralisation of all positively charged residues in IIS4 produced a functional channel (IIS4 N), while replacement of the charged residues in IS4, IIIS4 and IVS4 segments resulted in nonfunctional channels. The IIS4 N channel displayed activation kinetics similar to wild type. Mutations in a highly conserved structure motif on S6 segments ("GAGA ring": G432W in IS6, A780T in IIS6, G1193T in IIIS6 and A1503G in IVS6) induce strong left-shifted activation curves and decelerated channel deactivation kinetics. When IIS4 N was combined with these mutations, the activation curves were shifted back towards wild type and current kinetics were accelerated. In contrast, 12 other mutations adjacent to the GAGA ring in IS6-IVS6, which also affect activation gating, were not rescued by IIS4 N. Thus, the rescue of gating distortions in segments IS6-IVS6 by IIS4 N is highly position-specific. Thermodynamic cycle analysis supports the hypothesis that IIS4 is energetically coupled with the distantly located GAGA residues. We speculate that conformational changes caused by neutralisation of IIS4 are not restricted to domain II (IIS6) but are transmitted to gating structures in domains I, III and IV via the GAGA ring.
Point mutations in pore-lining S6 segments of CaV1.2 shift the voltage dependence of activation i... more Point mutations in pore-lining S6 segments of CaV1.2 shift the voltage dependence of activation into the hyperpolarizing direction and significantly decelerate current activation and deactivation. Here, we analyze theses changes in channel gating in terms of a circular four-state model accounting for an activation R–A–O and a deactivation O–D–R pathway. Transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements. Voltage-independent pore openings and closures during activation (A–O) and deactivation (D–R) are described by rate constants α and β, and γ and δ, respectively. Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)). Estimated model parameters of 15 CaV1.2 constructs well describe the activation and dea...
Voltage dependence and kinetics of Ca V 1.2 activation are affected by structural changes in pore... more Voltage dependence and kinetics of Ca V 1.2 activation are affected by structural changes in pore-lining S6 segments of the ␣ 1-subunit. Significant effects are induced by either proline or threonine substitutions in the lower third of segment IIS6 ("bundle crossing region"), where S6 segments are likely to seal the channel in the closed conformation (Hohaus, A.
4 The abbreviations used are: GFP, green fluorescent protein; WT, wild-type; NaChBac, bacterial s... more 4 The abbreviations used are: GFP, green fluorescent protein; WT, wild-type; NaChBac, bacterial sodium channel; Bay K8644, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)pyridine-3-carboxylic acid methyl ester.
The inner cavity of the hERG potassium ion channel can accommodate large, structurally diverse co... more The inner cavity of the hERG potassium ion channel can accommodate large, structurally diverse compounds that can be trapped in the channel by closure of the activation gate. A small set of propafenone derivatives was synthesized, and both use-dependency and recovery from block were tested in order to gain insight into the behavior of these compounds with respect to trapping and non-trapping. Ligand-protein docking into homology models of the closed and open state of the hERG channel provides the first evidence for the molecular basis of drug trapping.
A channelopathy mutation in segment IIS6 of Ca V 1.4 (I745T) has been shown to cause severe visua... more A channelopathy mutation in segment IIS6 of Ca V 1.4 (I745T) has been shown to cause severe visual impairment by shifting the activation and inactivation curves to more hyperpolarized voltages and slowing activation and inactivation kinetics. A similar gating phenotype is caused by the corresponding mutation, I781T, in Ca V 1.2 (midpoint of activation curve (V 0.5) shifted to-37.7 ± 1.2 mV). We show here that wild-type gating can partially be restored by a helix stabilizing rescue mutation N785A. V 0.5 of I781T/N785A (V 0.5 =-21.5 ± 0.6 mV) was shifted back towards wild-type (V 0.5 =-9.9 ± 1.1 mV). Homology models developed in our group (see accompanying article for details) were used to perform Molecular Dynamics-simulations (MD-simulations) on wild-type and mutant channels. Systematic changes in segment IIIS6 (M1187-F1194) and in helix IIS6 (N785-L786) were studied. The simulated structural changes in S6 segments of I781T/N785A were less pronounced than in I781T. A delicate balance between helix flexibility and stability enabling the formation of hydrophobic seals at the inner channel mouth appears to be important for wild-type Ca V 1.2 gating. Our study illustrates that effects of mutations in the lower part of IIS6 may not be localized to the residue or even segment being mutated, but may affect conformations of interacting segments.
Ca 2þ /calmodulin-dependent inactivation (CDI) of Ca V channels plays a crucial role in the homeo... more Ca 2þ /calmodulin-dependent inactivation (CDI) of Ca V channels plays a crucial role in the homeostasis of intracellular Ca 2þ , and serves as an ideal prototype for investigating Ca 2þ feedback regulation within biological systems. The prevailing view of CDI mechanism is that Ca 2þ-free calmodulin (apoCaM) preassociates with the IQ domain of the proximal carboxy terminus of channels, and this 'resident' calmodulin (CaM) acts as Ca 2þ sensor to somehow trigger CDI upon Ca 2þ binding. The downstream CaM/channel configurations leading to CDI are complex and under investigation at present. Contrasting with this complexity, Ca V 1.3 and Ca V 1.4 feature a long-carboxy-tail splice variant that minimizes CDI by a beautifully simple mechanism. We recently combined
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