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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 affects CaV2.1 channel inactivation properties.(A) Representative current traces illustrating the slower inactivation kinetics for ΔF1502 CaV2.1 channels (red trace) when compared to WT channels (blue trace), in response to a 3 s depolarizing pulse to +20 mV. (B) Average τinactivation values of Ca2+ currents through WT (open bar, n = 10) and ΔF1502 (filled bar, n = 8) CaV2.1 channels, elicited as indicated in panel (A). (C) Similar time course of Ca2+ current recovery from inactivation for WT and ΔF1502 CaV2.1 channels. Average τ of current recovery from inactivation obtained after fitting the data to a single exponential (solid color lines), were (in s): WT (open circles, n = 7) 15.5 ± 1.1; ΔF1502 (filled circles, n = 5) 16.9 ± 2.1 (P = 0.5, Student’s t test). (D, E) Steady-state inactivation of WT and ΔF1502 CaV2.1 channels. Amplitudes of currents elicited by test pulses to +20 mV (for WT channels) or -5 mV (for ΔF1502 channels) were normalized to the current obtained after a 30 s prepulse to -80 mV and fitted by a single Boltzmann function (solid color traces) (see Materials and Methods, Eq 2). Average V1/2 inact and kinact values were (in mV): WT (open circles, n = 19) -32.2 ± 2.1 and -5.3 ± 0.3; ΔF1502 (filled circles, n = 12) -60.7 ± 1 and -5.2 ± 0.8, respectively. No significant difference was found for kinact values (P = 0.89, Mann-Whitney test).
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pone.0146035.g005: ΔF1502 affects CaV2.1 channel inactivation properties.(A) Representative current traces illustrating the slower inactivation kinetics for ΔF1502 CaV2.1 channels (red trace) when compared to WT channels (blue trace), in response to a 3 s depolarizing pulse to +20 mV. (B) Average τinactivation values of Ca2+ currents through WT (open bar, n = 10) and ΔF1502 (filled bar, n = 8) CaV2.1 channels, elicited as indicated in panel (A). (C) Similar time course of Ca2+ current recovery from inactivation for WT and ΔF1502 CaV2.1 channels. Average τ of current recovery from inactivation obtained after fitting the data to a single exponential (solid color lines), were (in s): WT (open circles, n = 7) 15.5 ± 1.1; ΔF1502 (filled circles, n = 5) 16.9 ± 2.1 (P = 0.5, Student’s t test). (D, E) Steady-state inactivation of WT and ΔF1502 CaV2.1 channels. Amplitudes of currents elicited by test pulses to +20 mV (for WT channels) or -5 mV (for ΔF1502 channels) were normalized to the current obtained after a 30 s prepulse to -80 mV and fitted by a single Boltzmann function (solid color traces) (see Materials and Methods, Eq 2). Average V1/2 inact and kinact values were (in mV): WT (open circles, n = 19) -32.2 ± 2.1 and -5.3 ± 0.3; ΔF1502 (filled circles, n = 12) -60.7 ± 1 and -5.2 ± 0.8, respectively. No significant difference was found for kinact values (P = 0.89, Mann-Whitney test).

Mentions: Next, we studied whether ΔF1502 affects the time course of channel inactivation by analyzing the Ca2+ current decay during a 3-s test pulse elicited from a holding potential of -80 mV to +20 mV (Fig 5A). We found that inactivation kinetic for ΔF1502 CaV2.1 Ca2+ currents was significantly slower (τinactivation = 397.3 ± 37.1 ms, n = 8) than for WT currents (τinactivation = 121.3 ± 17.4 ms, n = 10) (Fig 5B, P < 0.0001, Student’s t test). However, the rate of recovery from inactivation was unaffected in ΔF1502 channels (Fig 5C, P = 0.5, Student’s t test). The half-maximal voltage for steady-state inactivation (V1/2 inact) induced by 30s conditioning prepulses between -80 and +5 mV was greatly left-shifted (~ 28.5 mV) in ΔF1502 channels (P < 0.0001, Student’s t test), without significant change in the steepness of the inactivation curve (symbolized by kinact) (P = 0.89, Mann-Whitney test) (Fig 5D and 5E).


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 affects CaV2.1 channel inactivation properties.(A) Representative current traces illustrating the slower inactivation kinetics for ΔF1502 CaV2.1 channels (red trace) when compared to WT channels (blue trace), in response to a 3 s depolarizing pulse to +20 mV. (B) Average τinactivation values of Ca2+ currents through WT (open bar, n = 10) and ΔF1502 (filled bar, n = 8) CaV2.1 channels, elicited as indicated in panel (A). (C) Similar time course of Ca2+ current recovery from inactivation for WT and ΔF1502 CaV2.1 channels. Average τ of current recovery from inactivation obtained after fitting the data to a single exponential (solid color lines), were (in s): WT (open circles, n = 7) 15.5 ± 1.1; ΔF1502 (filled circles, n = 5) 16.9 ± 2.1 (P = 0.5, Student’s t test). (D, E) Steady-state inactivation of WT and ΔF1502 CaV2.1 channels. Amplitudes of currents elicited by test pulses to +20 mV (for WT channels) or -5 mV (for ΔF1502 channels) were normalized to the current obtained after a 30 s prepulse to -80 mV and fitted by a single Boltzmann function (solid color traces) (see Materials and Methods, Eq 2). Average V1/2 inact and kinact values were (in mV): WT (open circles, n = 19) -32.2 ± 2.1 and -5.3 ± 0.3; ΔF1502 (filled circles, n = 12) -60.7 ± 1 and -5.2 ± 0.8, respectively. No significant difference was found for kinact values (P = 0.89, Mann-Whitney test).
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Related In: Results  -  Collection

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

pone.0146035.g005: ΔF1502 affects CaV2.1 channel inactivation properties.(A) Representative current traces illustrating the slower inactivation kinetics for ΔF1502 CaV2.1 channels (red trace) when compared to WT channels (blue trace), in response to a 3 s depolarizing pulse to +20 mV. (B) Average τinactivation values of Ca2+ currents through WT (open bar, n = 10) and ΔF1502 (filled bar, n = 8) CaV2.1 channels, elicited as indicated in panel (A). (C) Similar time course of Ca2+ current recovery from inactivation for WT and ΔF1502 CaV2.1 channels. Average τ of current recovery from inactivation obtained after fitting the data to a single exponential (solid color lines), were (in s): WT (open circles, n = 7) 15.5 ± 1.1; ΔF1502 (filled circles, n = 5) 16.9 ± 2.1 (P = 0.5, Student’s t test). (D, E) Steady-state inactivation of WT and ΔF1502 CaV2.1 channels. Amplitudes of currents elicited by test pulses to +20 mV (for WT channels) or -5 mV (for ΔF1502 channels) were normalized to the current obtained after a 30 s prepulse to -80 mV and fitted by a single Boltzmann function (solid color traces) (see Materials and Methods, Eq 2). Average V1/2 inact and kinact values were (in mV): WT (open circles, n = 19) -32.2 ± 2.1 and -5.3 ± 0.3; ΔF1502 (filled circles, n = 12) -60.7 ± 1 and -5.2 ± 0.8, respectively. No significant difference was found for kinact values (P = 0.89, Mann-Whitney test).
Mentions: Next, we studied whether ΔF1502 affects the time course of channel inactivation by analyzing the Ca2+ current decay during a 3-s test pulse elicited from a holding potential of -80 mV to +20 mV (Fig 5A). We found that inactivation kinetic for ΔF1502 CaV2.1 Ca2+ currents was significantly slower (τinactivation = 397.3 ± 37.1 ms, n = 8) than for WT currents (τinactivation = 121.3 ± 17.4 ms, n = 10) (Fig 5B, P < 0.0001, Student’s t test). However, the rate of recovery from inactivation was unaffected in ΔF1502 channels (Fig 5C, P = 0.5, Student’s t test). The half-maximal voltage for steady-state inactivation (V1/2 inact) induced by 30s conditioning prepulses between -80 and +5 mV was greatly left-shifted (~ 28.5 mV) in ΔF1502 channels (P < 0.0001, Student’s t test), without significant change in the steepness of the inactivation curve (symbolized by kinact) (P = 0.89, Mann-Whitney test) (Fig 5D and 5E).

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