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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.

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ΔF1502 effects on Ca2+ influx evoked by single action potential-like waveforms (APWs).(A) Average Ca2+ current traces evoked by APWs of different durations (fast (left panels), medium (central panels) and slow (right panels) (see Materials and Methods for details) obtained from tsA-201 HEK cells expressing WT (blue traces) or ΔF1502 (red traces) CaV2.1 channels. Dotted lines stand for the zero current level. (B) Average data for peak Ca2+ (ICa2+) current density (top panel), normalized Ca2+ influx (QCa2+) (second panel), time to peak (third panel), and time for Ca2+ entry (bottom panel) in response to APWs of different durations obtained from cells expressing WT (blue bars, n = 12–14) or ΔF1502 (red bars, n = 8) CaV2.1 channels (*P < 0.05, **P < 0.001 and ***P < 0.0001 when compared to WT).
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pone.0146035.g006: ΔF1502 effects on Ca2+ influx evoked by single action potential-like waveforms (APWs).(A) Average Ca2+ current traces evoked by APWs of different durations (fast (left panels), medium (central panels) and slow (right panels) (see Materials and Methods for details) obtained from tsA-201 HEK cells expressing WT (blue traces) or ΔF1502 (red traces) CaV2.1 channels. Dotted lines stand for the zero current level. (B) Average data for peak Ca2+ (ICa2+) current density (top panel), normalized Ca2+ influx (QCa2+) (second panel), time to peak (third panel), and time for Ca2+ entry (bottom panel) in response to APWs of different durations obtained from cells expressing WT (blue bars, n = 12–14) or ΔF1502 (red bars, n = 8) CaV2.1 channels (*P < 0.05, **P < 0.001 and ***P < 0.0001 when compared to WT).

Mentions: To better understand the physiological impact of ΔF1502 effects on CaV2.1 channel activity, we measured the Ca2+ influx through WT and mutant channels elicited by single action potential-like waveforms (APWs) of different durations (fast, medium and slow; Fig 6A, top; for further details see Materials and Methods [13,57]). In spite of the significant reduction in maximal Ca2+ current density produced by ΔF1502 (in this set of experiments maximal Ca2+ current density through ΔF1502 channels in response to a 20 ms depolarizing pulse was ~ 50% smaller when compared to WT current, S1 Fig, left panel), no significant changes among cells expressing either WT or ΔF1502 channels were observed regarding the peak Ca2+ current density elicited by the different APWs (Fig 6A and 6B, top panel). Besides, and more important, total Ca2+ influx (QCa2+) in response to APWs was not reduced by ΔF1502. Not only that, but the amount of Ca2+ that entered into the cell in response to fast and medium APWs was significantly higher for cells expressing the CaV2.1 mutant channel (Fig 6A and 6B, second panel), most probably due to the ~ 18.7 mV ΔF1502-induced left-shit in the activation curve observed in this set of cells (S1 Fig, right panel). This, in combination with the effects that ΔF1502 exerts on channel activation and deactivation kinetics, may also explain another two facts: the time required to reach the peak current density in response to all APWs was always significantly higher in cells expressing the mutant CaV2.1 channel (Fig 6A and 6B, third panel); and the time of Ca2+ entry in response to fast and medium APWs was significantly higher in cells expressing the mutant CaV2.1 channel (Fig 6A and 6B, bottom panel).


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 single action potential-like waveforms (APWs).(A) Average Ca2+ current traces evoked by APWs of different durations (fast (left panels), medium (central panels) and slow (right panels) (see Materials and Methods for details) obtained from tsA-201 HEK cells expressing WT (blue traces) or ΔF1502 (red traces) CaV2.1 channels. Dotted lines stand for the zero current level. (B) Average data for peak Ca2+ (ICa2+) current density (top panel), normalized Ca2+ influx (QCa2+) (second panel), time to peak (third panel), and time for Ca2+ entry (bottom panel) in response to APWs of different durations obtained from cells expressing WT (blue bars, n = 12–14) or ΔF1502 (red bars, n = 8) CaV2.1 channels (*P < 0.05, **P < 0.001 and ***P < 0.0001 when compared to WT).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0146035.g006: ΔF1502 effects on Ca2+ influx evoked by single action potential-like waveforms (APWs).(A) Average Ca2+ current traces evoked by APWs of different durations (fast (left panels), medium (central panels) and slow (right panels) (see Materials and Methods for details) obtained from tsA-201 HEK cells expressing WT (blue traces) or ΔF1502 (red traces) CaV2.1 channels. Dotted lines stand for the zero current level. (B) Average data for peak Ca2+ (ICa2+) current density (top panel), normalized Ca2+ influx (QCa2+) (second panel), time to peak (third panel), and time for Ca2+ entry (bottom panel) in response to APWs of different durations obtained from cells expressing WT (blue bars, n = 12–14) or ΔF1502 (red bars, n = 8) CaV2.1 channels (*P < 0.05, **P < 0.001 and ***P < 0.0001 when compared to WT).
Mentions: To better understand the physiological impact of ΔF1502 effects on CaV2.1 channel activity, we measured the Ca2+ influx through WT and mutant channels elicited by single action potential-like waveforms (APWs) of different durations (fast, medium and slow; Fig 6A, top; for further details see Materials and Methods [13,57]). In spite of the significant reduction in maximal Ca2+ current density produced by ΔF1502 (in this set of experiments maximal Ca2+ current density through ΔF1502 channels in response to a 20 ms depolarizing pulse was ~ 50% smaller when compared to WT current, S1 Fig, left panel), no significant changes among cells expressing either WT or ΔF1502 channels were observed regarding the peak Ca2+ current density elicited by the different APWs (Fig 6A and 6B, top panel). Besides, and more important, total Ca2+ influx (QCa2+) in response to APWs was not reduced by ΔF1502. Not only that, but the amount of Ca2+ that entered into the cell in response to fast and medium APWs was significantly higher for cells expressing the CaV2.1 mutant channel (Fig 6A and 6B, second panel), most probably due to the ~ 18.7 mV ΔF1502-induced left-shit in the activation curve observed in this set of cells (S1 Fig, right panel). This, in combination with the effects that ΔF1502 exerts on channel activation and deactivation kinetics, may also explain another two facts: the time required to reach the peak current density in response to all APWs was always significantly higher in cells expressing the mutant CaV2.1 channel (Fig 6A and 6B, third panel); and the time of Ca2+ entry in response to fast and medium APWs was significantly higher in cells expressing the mutant CaV2.1 channel (Fig 6A and 6B, bottom panel).

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