Piezo2 channel regulates RhoA and actin cytoskeleton to promote cell mechanobiological responses

Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2

Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine: clinical, genetic and functional studies

Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A-p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2-p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing

A Single Amino Acid Deletion (ΔF1502) in the S6 Segment of Ca2.1 Domain III Associated with Congenital Ataxia Increases Channel Activity and Promotes Ca 2+ Influx

Mutations in the CACNA1A gene, encoding the pore-forming Ca2.1 (P/Q-type) channel α 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 α domain III (ΔF1502). Functional studies of ΔF1502 Ca2.1 channels, expressed in Xenopus oocytes, using the non-physiological Ba 2+ 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 α mutation, detected by whole-exome sequencing, and analyze its functional consequences on Ca2.1 human channels heterologously expressed in mammalian tsA-201 HEK cells, using the physiological permeant ion Ca 2+. ΔF1502 strongly decreases the voltage threshold for channel activation (by ~ 21 mV), allowing significantly higher Ca 2+ current densities in a range of depolarized voltages with physiological relevance in neurons, even though maximal Ca 2+ current density through ΔF1502 Ca2.1 channels is 60% lower than through wild-type channels. ΔF1502 accelerates activation kinetics and slows deactivation kinetics of Ca2.1 within a wide range of voltage depolarization. ΔF1502 also slowed Ca2.1 inactivation kinetic and shifted the inactivation curve to hyperpolarized potentials (by ~ 28 mV). ΔF1502 effects on Ca2.1 activation and deactivation properties seem to be of high physiological relevance. Thus, ΔF1502 strongly promotes Ca 2+ 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 Ca2.1 mutations in congenital ataxia, a neurodevelopmental disorder at the severe-most end of CACNA1A -associated phenotypic spectru

<i>De novo</i> heterozygous <i>CACNA1A</i> deletion in congenital ataxia with cerebellar atrophy.

<p>(A) Pedigree of the affected individual carrying the <i>de novo</i> heterozygous ΔF1502 mutation. White symbols denote healthy individuals and grey, congenital ataxia. (B) Electropherograms showing the deleted nucleotides (bracket) (NM_001127221.1-transcript variant 3:c.4503-4505delCTT) leading to a F1052 deletion (NP_001120693.1). Note the double wild-type (WT) and mutant (ΔF1502) sequence in the patient’s electropherogram (heterozygous mutation carrier).</p

Evolutionary conservation of the F1502 residue and predicted location at the channel pore.

<p>(A) Sequence alignment of individual S6 segments at domains I to IV (DI-DIV) of human Ca_V2.x channel α₁ subunits (P/Q type Ca_V2.1; N-type Ca_V2.2; R-type Ca_V2.3), human Ca_V1.x (L-type) channel α₁ subunits, and the bacterial sodium channel Na_VAb (top); sequence alignment of S6-DIII of Ca_V2.1 channels from different species (as indicated). The three Phenylalanine’s group (in red) is conserved in the human Ca_V2.1 channel α_1A subunit, where F1502 is located at the third position. This particular amino acid residue is only conserved in S6-DIII of Ca_V2 type channels. The phenylalanine’s group is totally conserved in S6-DIII of Ca_V2.1 channels from different species. The alignments were performed with T-Coffee (T-Coffee). (B,C,D) Location of the F1502 homologous methionine residue (M209), using the Na_VAb structure as a model (PDB 4EKW). A methionine residue is also present at the F1502 position in L-type channels. The side view (B) show a red highlighted M209 residue in Na_VAb, which lines the inner pore vestibule of the channel. A view from the cytoplasm looking up through the channel pore show the arrangement of M209 residue in the four Na_VAb subunits (C), and a zoom of the pore region from the same view is shown in (D). Images were generated using UCSF Chimera package. Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146035#pone.0146035.ref079" target="_blank">79</a>].</p

Brain MRI of the proband at the age of 14 months (A), 28 months (B), and 4 and a half years (C,D).

<p>After the initial normal findings (A), note the progressive cerebellar atrophy mainly involving the complete vermis (indicated by the arrows in B, C). The hemispheres, displaying prominence of the cerebellar folia, were eventually affected (D).</p

ΔF1502 induces a gain-of-function in the heterologously expressed Ca_V2.1 channel by affecting its activation and deactivation properties.

<p>(A) Representative current traces elicited by 20 ms depolarizing pulses from -80 mV to the indicated voltages (inset), illustrating the difference in voltage-dependence and activation kinetics between wild-type (WT) (left) and ΔF1502 (right) Ca_V2.1 channels. Dotted lines indicate the zero current level. (B) Representative current traces showing distinct deactivation kinetics of WT (left) and ΔF1502 (right) Ca_V2.1 channels, obtained by hyperpolarizing the cells during 30 ms at the indicated voltages (inset) following a 20 ms depolarizing pulse to +20 mV (for WT channels) or -5 mV (for ΔF1502 channels). The zero current level is indicated by dotted lines. (C) Average current density-voltage relationships (left) and normalized I-V curves (right) for WT (open circles, n = 27) and ΔF1502 (filled circles, n = 19) Ca_V2.1 channels expressed in tsA-201 HEK cells. Red box indicates the voltage range at which peak Ca²⁺ current densities through ΔF1502 channels exceed those produced by WT channels. Average V_{1/2 act}, k_act and V_rev values were (in mV): WT (open circles, n = 27) 3.8 ± 0.6, 3.5 ± 0.15 and 62.4 ± 1.4; ΔF1502 (filled circles, n = 19) -17.1 ± 0.9, 4.4 ± 0.19 and 51.6 ± 2.2, respectively. Average activation (D) and deactivation (E) kinetics of WT (open circles) and ΔF1502 Ca_V2.1 Ca²⁺ currents (filled circles) at the indicated voltages.</p

ΔF1502 effects on Ca²⁺ influx evoked by a 42 Hz train of 2 ms action potential-like waveforms (APWs).

<p>(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) Ca_V2.1 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 Ca_V2.1 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 <i>t</i> test) and the significant left-shift induced by ΔF1502 on the Ca_V2.1 voltage-dependent activation is also noticed (right panel: WT V_{1/2 act} = 2.32 ± 1.18 mV (n = 10) <i>versus</i> ΔF1502 V_{1/2 act} = -17.74 ± 0.35 mV (n = 11), P < 0.0001, Student’s <i>t</i> test). (B) Representative Ca²⁺ current traces evoked by every 200^th pulse of a 42 Hz train of medium (2 ms) APWs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146035#sec002" target="_blank">Materials and Methods</a> for details) obtained from two tsA-201 HEK cells expressing either WT (left) or ΔF1502 (right) Ca_V2.1 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 Ca_V2.1 channels are -115.28 pA/pF and -52.27 pA/pF, respectively; V_{1/2 act} values for WT and ΔF1502 Ca_V2.1 channels are 2.52 mV and -17.23 mV, respectively). (C) Average data for Ca²⁺ influx normalized by cell size (Q_Ca²⁺) in response to every 5^th 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) Ca_V2.1 channels.</p

Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine : clinical, genetic, and functional studies

Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A -p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2 -p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing

Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine: clinical, genetic and functional studies

Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A-p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2-p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing