Limits...
A Single Amino Acid Deletion (ΔF1502) in the S6 Segment of CaV2.1 Domain III Associated with Congenital Ataxia Increases Channel Activity and Promotes Ca2+ Influx.

Bahamonde MI, Serra SA, Drechsel O, Rahman R, Marcé-Grau A, Prieto M, Ossowski S, Macaya A, Fernández-Fernández JM - PLoS ONE (2015)

Bottom Line: ΔF1502 strongly decreases the voltage threshold for channel activation (by ~ 21 mV), allowing significantly higher Ca2+ current densities in a range of depolarized voltages with physiological relevance in neurons, even though maximal Ca2+ current density through ΔF1502 CaV2.1 channels is 60% lower than through wild-type channels.ΔF1502 effects on CaV2.1 activation and deactivation properties seem to be of high physiological relevance.Thus, ΔF1502 strongly promotes Ca2+ influx in response to either single or trains of action potential-like waveforms of different durations.

View Article: PubMed Central - PubMed

Affiliation: Laboratori de Fisiologia Molecular i Canalopaties, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.

ABSTRACT
Mutations in the CACNA1A gene, encoding the pore-forming CaV2.1 (P/Q-type) channel α1A subunit, result in heterogeneous human neurological disorders, including familial and sporadic hemiplegic migraine along with episodic and progressive forms of ataxia. Hemiplegic Migraine (HM) mutations induce gain-of-channel function, mainly by shifting channel activation to lower voltages, whereas ataxia mutations mostly produce loss-of-channel function. However, some HM-linked gain-of-function mutations are also associated to congenital ataxia and/or cerebellar atrophy, including the deletion of a highly conserved phenylalanine located at the S6 pore region of α1A domain III (ΔF1502). Functional studies of ΔF1502 CaV2.1 channels, expressed in Xenopus oocytes, using the non-physiological Ba2+ as the charge carrier have only revealed discrete alterations in channel function of unclear pathophysiological relevance. Here, we report a second case of congenital ataxia linked to the ΔF1502 α1A mutation, detected by whole-exome sequencing, and analyze its functional consequences on CaV2.1 human channels heterologously expressed in mammalian tsA-201 HEK cells, using the physiological permeant ion Ca2+. ΔF1502 strongly decreases the voltage threshold for channel activation (by ~ 21 mV), allowing significantly higher Ca2+ current densities in a range of depolarized voltages with physiological relevance in neurons, even though maximal Ca2+ current density through ΔF1502 CaV2.1 channels is 60% lower than through wild-type channels. ΔF1502 accelerates activation kinetics and slows deactivation kinetics of CaV2.1 within a wide range of voltage depolarization. ΔF1502 also slowed CaV2.1 inactivation kinetic and shifted the inactivation curve to hyperpolarized potentials (by ~ 28 mV). ΔF1502 effects on CaV2.1 activation and deactivation properties seem to be of high physiological relevance. Thus, ΔF1502 strongly promotes Ca2+ influx in response to either single or trains of action potential-like waveforms of different durations. Our observations support a causative role of gain-of-function CaV2.1 mutations in congenital ataxia, a neurodevelopmental disorder at the severe-most end of CACNA1A-associated phenotypic spectrum.

Show MeSH

Related in: MedlinePlus

ΔF1502 effects on Ca2+ influx evoked by a 42 Hz train of 2 ms action potential-like waveforms (APWs).(A) Average current density-voltage relationships (left) and normalized I-V curves (right) for WT (open circles, n = 10) and ΔF1502 (filled circles, n = 11) CaV2.1 channels expressed in tsA-201 HEK cells, before stimulation with a 42 Hz train of 2 ms APWs. In this series of experiments, maximal Ca2+ current density through CaV2.1 channels is still significantly reduced by ΔF1502 (left panel: from -94.26 ± 18.9 pA/pF (for WT, n = 10) to -47.76 ± 5.7 pA/pF (for ΔF1502, n = 11), P < 0.05, Student’s t test) and the significant left-shift induced by ΔF1502 on the CaV2.1 voltage-dependent activation is also noticed (right panel: WT V1/2 act = 2.32 ± 1.18 mV (n = 10) versus ΔF1502 V1/2 act = -17.74 ± 0.35 mV (n = 11), P < 0.0001, Student’s t test). (B) Representative Ca2+ current traces evoked by every 200th pulse of a 42 Hz train of medium (2 ms) APWs (see Materials and Methods for details) obtained from two tsA-201 HEK cells expressing either WT (left) or ΔF1502 (right) CaV2.1 channels. Dotted lines stand for the zero current level. The corresponding current density-voltage relationships (left) and normalized I-V curves (right), obtained from these two cells before stimulation with a 42 Hz train of 2 ms APWs, are shown at the bottom (maximal Ca2+ current density through WT and ΔF1502 CaV2.1 channels are -115.28 pA/pF and -52.27 pA/pF, respectively; V1/2 act values for WT and ΔF1502 CaV2.1 channels are 2.52 mV and -17.23 mV, respectively). (C) Average data for Ca2+ influx normalized by cell size (QCa2+) in response to every 5th pulse of a 42 Hz train of medium (2 ms) APWs, obtained from cells expressing WT (blue symbols, n = 10) or ΔF1502 (red symbols, n = 11) CaV2.1 channels.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4696675&req=5

pone.0146035.g008: ΔF1502 effects on Ca2+ influx evoked by a 42 Hz train of 2 ms action potential-like waveforms (APWs).(A) Average current density-voltage relationships (left) and normalized I-V curves (right) for WT (open circles, n = 10) and ΔF1502 (filled circles, n = 11) CaV2.1 channels expressed in tsA-201 HEK cells, before stimulation with a 42 Hz train of 2 ms APWs. In this series of experiments, maximal Ca2+ current density through CaV2.1 channels is still significantly reduced by ΔF1502 (left panel: from -94.26 ± 18.9 pA/pF (for WT, n = 10) to -47.76 ± 5.7 pA/pF (for ΔF1502, n = 11), P < 0.05, Student’s t test) and the significant left-shift induced by ΔF1502 on the CaV2.1 voltage-dependent activation is also noticed (right panel: WT V1/2 act = 2.32 ± 1.18 mV (n = 10) versus ΔF1502 V1/2 act = -17.74 ± 0.35 mV (n = 11), P < 0.0001, Student’s t test). (B) Representative Ca2+ current traces evoked by every 200th pulse of a 42 Hz train of medium (2 ms) APWs (see Materials and Methods for details) obtained from two tsA-201 HEK cells expressing either WT (left) or ΔF1502 (right) CaV2.1 channels. Dotted lines stand for the zero current level. The corresponding current density-voltage relationships (left) and normalized I-V curves (right), obtained from these two cells before stimulation with a 42 Hz train of 2 ms APWs, are shown at the bottom (maximal Ca2+ current density through WT and ΔF1502 CaV2.1 channels are -115.28 pA/pF and -52.27 pA/pF, respectively; V1/2 act values for WT and ΔF1502 CaV2.1 channels are 2.52 mV and -17.23 mV, respectively). (C) Average data for Ca2+ influx normalized by cell size (QCa2+) in response to every 5th pulse of a 42 Hz train of medium (2 ms) APWs, obtained from cells expressing WT (blue symbols, n = 10) or ΔF1502 (red symbols, n = 11) CaV2.1 channels.

Mentions: As ΔF1502 strongly promotes CaV2.1 channel steady-state inactivation by voltage in response to prolonged depolarization (Fig 5D and 5E), we also evaluated if this last action is relevant for Ca2+ influx through the channel under physiological conditions. In vivo, neurons do not experience long depolarization to induce CaV2.1 steady-state inactivation. Instead, this effect on CaV2.1 channel activity is mimicked by the occurrence of more physiological stimuli, such as trains of action potentials causing repetitive, brief depolarization [59]. Therefore, given that total Ca2+ influx (QCa2+) in response to single fast and medium APWs was favored by ΔF1502, we studied the effect of the mutation on QCa2+ elicited by trains of fast and medium APWs applied at relatively high frequency in two new sets of experiments, where it can be observed again the reduction in maximal Ca2+ current density (by ~ 50%) and the shift of the current activation curve to lower voltages (~ 20 mV) induced by ΔF1502 (Figs 7A and 8A). The application of a train of fast APWs (1 ms duration, with maximal depolarization to +30 mV from a holding potential of -80 mV, applied at 50 Hz for 20 seconds) did not cause a significant reduction in Ca2+ influx through WT CaV2.1 channels (QCa2+ produced by the first APW applied was 1.94 ± 0.3 fC/pF (n = 9) and at the end of the train the observed QCa2+ value was 1.76 ± 0.3 fC/pF (n = 9), P = 0.23, paired Student’s t test). However, and according to the effect of ΔF1502 on CaV2.1 channel steady-state inactivation, the same train of fast APWs produced a small, but significant, reduction (by ~ 19%) in Ca2+ influx through ΔF1502 CaV2.1 channels (from 5.76 ± 0.7 fC/pF to 4.69 ± 0.6 fC/pF (n = 7), P < 0.01, paired Student’s t test) (Fig 7B and 7C). Nevertheless, in spite of this Ca2+ entry reduction along the train produced by ΔF1502, QCa2+ after each APW stimulus was always higher for mutant channels (Fig 7B and 7C) and, therefore, accumulative Ca2+ influx through ΔF1502 CaV2.1 channels was still significantly higher than through WT channels (4.7 ± 0.4 pC/pF (n = 7) versus 1.9 ± 0.3 pC/pF (n = 9), P < 0.0001, Student’s t test). Similar conclusions can be drawn from the analysis of studies using trains of medium APWs (2 ms duration, with maximal depolarization to +30 mV from a holding potential of -80 mV, applied at 42 Hz for 23.81 seconds) (Fig 8). In this case, the train of APWs produced a significant reduction in Ca2+ influx through both, WT (from 9.27 ± 2.14 fC/pF to 6.97 ± 1.57 fC/pF (n = 10), P < 0.01, paired Student’s t test) and ΔF1502 (from 20.81 ± 1.9 fC/pF to 9.6 ± 1.3 fC/pF (n = 11), P < 0.0001, paired Student’s t test) CaV2.1 channels, which was significantly greater for mutant channels (54.65 ± 4.4% (n = 10) versus 23.69 ± 2.9% (n = 11), P < 0.0001, Student’s t test) (Fig 8B and 8C). Yet, accumulative Ca2+ influx through ΔF1502 CaV2.1 channels all along the train was significantly higher than through WT channels (13.4 ± 1.4 pC/pF (n = 11) versus 7.9 ± 1.8 pC/pF (n = 10), P < 0.05, Student’s t test) (Fig 8B and 8C).


A Single Amino Acid Deletion (ΔF1502) in the S6 Segment of CaV2.1 Domain III Associated with Congenital Ataxia Increases Channel Activity and Promotes Ca2+ Influx.

Bahamonde MI, Serra SA, Drechsel O, Rahman R, Marcé-Grau A, Prieto M, Ossowski S, Macaya A, Fernández-Fernández JM - PLoS ONE (2015)

ΔF1502 effects on Ca2+ influx evoked by a 42 Hz train of 2 ms action potential-like waveforms (APWs).(A) Average current density-voltage relationships (left) and normalized I-V curves (right) for WT (open circles, n = 10) and ΔF1502 (filled circles, n = 11) CaV2.1 channels expressed in tsA-201 HEK cells, before stimulation with a 42 Hz train of 2 ms APWs. In this series of experiments, maximal Ca2+ current density through CaV2.1 channels is still significantly reduced by ΔF1502 (left panel: from -94.26 ± 18.9 pA/pF (for WT, n = 10) to -47.76 ± 5.7 pA/pF (for ΔF1502, n = 11), P < 0.05, Student’s t test) and the significant left-shift induced by ΔF1502 on the CaV2.1 voltage-dependent activation is also noticed (right panel: WT V1/2 act = 2.32 ± 1.18 mV (n = 10) versus ΔF1502 V1/2 act = -17.74 ± 0.35 mV (n = 11), P < 0.0001, Student’s t test). (B) Representative Ca2+ current traces evoked by every 200th pulse of a 42 Hz train of medium (2 ms) APWs (see Materials and Methods for details) obtained from two tsA-201 HEK cells expressing either WT (left) or ΔF1502 (right) CaV2.1 channels. Dotted lines stand for the zero current level. The corresponding current density-voltage relationships (left) and normalized I-V curves (right), obtained from these two cells before stimulation with a 42 Hz train of 2 ms APWs, are shown at the bottom (maximal Ca2+ current density through WT and ΔF1502 CaV2.1 channels are -115.28 pA/pF and -52.27 pA/pF, respectively; V1/2 act values for WT and ΔF1502 CaV2.1 channels are 2.52 mV and -17.23 mV, respectively). (C) Average data for Ca2+ influx normalized by cell size (QCa2+) in response to every 5th pulse of a 42 Hz train of medium (2 ms) APWs, obtained from cells expressing WT (blue symbols, n = 10) or ΔF1502 (red symbols, n = 11) CaV2.1 channels.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696675&req=5

pone.0146035.g008: ΔF1502 effects on Ca2+ influx evoked by a 42 Hz train of 2 ms action potential-like waveforms (APWs).(A) Average current density-voltage relationships (left) and normalized I-V curves (right) for WT (open circles, n = 10) and ΔF1502 (filled circles, n = 11) CaV2.1 channels expressed in tsA-201 HEK cells, before stimulation with a 42 Hz train of 2 ms APWs. In this series of experiments, maximal Ca2+ current density through CaV2.1 channels is still significantly reduced by ΔF1502 (left panel: from -94.26 ± 18.9 pA/pF (for WT, n = 10) to -47.76 ± 5.7 pA/pF (for ΔF1502, n = 11), P < 0.05, Student’s t test) and the significant left-shift induced by ΔF1502 on the CaV2.1 voltage-dependent activation is also noticed (right panel: WT V1/2 act = 2.32 ± 1.18 mV (n = 10) versus ΔF1502 V1/2 act = -17.74 ± 0.35 mV (n = 11), P < 0.0001, Student’s t test). (B) Representative Ca2+ current traces evoked by every 200th pulse of a 42 Hz train of medium (2 ms) APWs (see Materials and Methods for details) obtained from two tsA-201 HEK cells expressing either WT (left) or ΔF1502 (right) CaV2.1 channels. Dotted lines stand for the zero current level. The corresponding current density-voltage relationships (left) and normalized I-V curves (right), obtained from these two cells before stimulation with a 42 Hz train of 2 ms APWs, are shown at the bottom (maximal Ca2+ current density through WT and ΔF1502 CaV2.1 channels are -115.28 pA/pF and -52.27 pA/pF, respectively; V1/2 act values for WT and ΔF1502 CaV2.1 channels are 2.52 mV and -17.23 mV, respectively). (C) Average data for Ca2+ influx normalized by cell size (QCa2+) in response to every 5th pulse of a 42 Hz train of medium (2 ms) APWs, obtained from cells expressing WT (blue symbols, n = 10) or ΔF1502 (red symbols, n = 11) CaV2.1 channels.
Mentions: As ΔF1502 strongly promotes CaV2.1 channel steady-state inactivation by voltage in response to prolonged depolarization (Fig 5D and 5E), we also evaluated if this last action is relevant for Ca2+ influx through the channel under physiological conditions. In vivo, neurons do not experience long depolarization to induce CaV2.1 steady-state inactivation. Instead, this effect on CaV2.1 channel activity is mimicked by the occurrence of more physiological stimuli, such as trains of action potentials causing repetitive, brief depolarization [59]. Therefore, given that total Ca2+ influx (QCa2+) in response to single fast and medium APWs was favored by ΔF1502, we studied the effect of the mutation on QCa2+ elicited by trains of fast and medium APWs applied at relatively high frequency in two new sets of experiments, where it can be observed again the reduction in maximal Ca2+ current density (by ~ 50%) and the shift of the current activation curve to lower voltages (~ 20 mV) induced by ΔF1502 (Figs 7A and 8A). The application of a train of fast APWs (1 ms duration, with maximal depolarization to +30 mV from a holding potential of -80 mV, applied at 50 Hz for 20 seconds) did not cause a significant reduction in Ca2+ influx through WT CaV2.1 channels (QCa2+ produced by the first APW applied was 1.94 ± 0.3 fC/pF (n = 9) and at the end of the train the observed QCa2+ value was 1.76 ± 0.3 fC/pF (n = 9), P = 0.23, paired Student’s t test). However, and according to the effect of ΔF1502 on CaV2.1 channel steady-state inactivation, the same train of fast APWs produced a small, but significant, reduction (by ~ 19%) in Ca2+ influx through ΔF1502 CaV2.1 channels (from 5.76 ± 0.7 fC/pF to 4.69 ± 0.6 fC/pF (n = 7), P < 0.01, paired Student’s t test) (Fig 7B and 7C). Nevertheless, in spite of this Ca2+ entry reduction along the train produced by ΔF1502, QCa2+ after each APW stimulus was always higher for mutant channels (Fig 7B and 7C) and, therefore, accumulative Ca2+ influx through ΔF1502 CaV2.1 channels was still significantly higher than through WT channels (4.7 ± 0.4 pC/pF (n = 7) versus 1.9 ± 0.3 pC/pF (n = 9), P < 0.0001, Student’s t test). Similar conclusions can be drawn from the analysis of studies using trains of medium APWs (2 ms duration, with maximal depolarization to +30 mV from a holding potential of -80 mV, applied at 42 Hz for 23.81 seconds) (Fig 8). In this case, the train of APWs produced a significant reduction in Ca2+ influx through both, WT (from 9.27 ± 2.14 fC/pF to 6.97 ± 1.57 fC/pF (n = 10), P < 0.01, paired Student’s t test) and ΔF1502 (from 20.81 ± 1.9 fC/pF to 9.6 ± 1.3 fC/pF (n = 11), P < 0.0001, paired Student’s t test) CaV2.1 channels, which was significantly greater for mutant channels (54.65 ± 4.4% (n = 10) versus 23.69 ± 2.9% (n = 11), P < 0.0001, Student’s t test) (Fig 8B and 8C). Yet, accumulative Ca2+ influx through ΔF1502 CaV2.1 channels all along the train was significantly higher than through WT channels (13.4 ± 1.4 pC/pF (n = 11) versus 7.9 ± 1.8 pC/pF (n = 10), P < 0.05, Student’s t test) (Fig 8B and 8C).

Bottom Line: ΔF1502 strongly decreases the voltage threshold for channel activation (by ~ 21 mV), allowing significantly higher Ca2+ current densities in a range of depolarized voltages with physiological relevance in neurons, even though maximal Ca2+ current density through ΔF1502 CaV2.1 channels is 60% lower than through wild-type channels.ΔF1502 effects on CaV2.1 activation and deactivation properties seem to be of high physiological relevance.Thus, ΔF1502 strongly promotes Ca2+ influx in response to either single or trains of action potential-like waveforms of different durations.

View Article: PubMed Central - PubMed

Affiliation: Laboratori de Fisiologia Molecular i Canalopaties, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.

ABSTRACT
Mutations in the CACNA1A gene, encoding the pore-forming CaV2.1 (P/Q-type) channel α1A subunit, result in heterogeneous human neurological disorders, including familial and sporadic hemiplegic migraine along with episodic and progressive forms of ataxia. Hemiplegic Migraine (HM) mutations induce gain-of-channel function, mainly by shifting channel activation to lower voltages, whereas ataxia mutations mostly produce loss-of-channel function. However, some HM-linked gain-of-function mutations are also associated to congenital ataxia and/or cerebellar atrophy, including the deletion of a highly conserved phenylalanine located at the S6 pore region of α1A domain III (ΔF1502). Functional studies of ΔF1502 CaV2.1 channels, expressed in Xenopus oocytes, using the non-physiological Ba2+ as the charge carrier have only revealed discrete alterations in channel function of unclear pathophysiological relevance. Here, we report a second case of congenital ataxia linked to the ΔF1502 α1A mutation, detected by whole-exome sequencing, and analyze its functional consequences on CaV2.1 human channels heterologously expressed in mammalian tsA-201 HEK cells, using the physiological permeant ion Ca2+. ΔF1502 strongly decreases the voltage threshold for channel activation (by ~ 21 mV), allowing significantly higher Ca2+ current densities in a range of depolarized voltages with physiological relevance in neurons, even though maximal Ca2+ current density through ΔF1502 CaV2.1 channels is 60% lower than through wild-type channels. ΔF1502 accelerates activation kinetics and slows deactivation kinetics of CaV2.1 within a wide range of voltage depolarization. ΔF1502 also slowed CaV2.1 inactivation kinetic and shifted the inactivation curve to hyperpolarized potentials (by ~ 28 mV). ΔF1502 effects on CaV2.1 activation and deactivation properties seem to be of high physiological relevance. Thus, ΔF1502 strongly promotes Ca2+ influx in response to either single or trains of action potential-like waveforms of different durations. Our observations support a causative role of gain-of-function CaV2.1 mutations in congenital ataxia, a neurodevelopmental disorder at the severe-most end of CACNA1A-associated phenotypic spectrum.

Show MeSH
Related in: MedlinePlus