1. Evolutionary Biology
  2. Structural Biology and Molecular Biophysics

OSCA/TMEM63 are an Evolutionarily Conserved Family of Mechanically Activated Ion Channels

  1. Swetha E Murthy  Is a corresponding author
  2. Adrienne E Dubin  Is a corresponding author
  3. Tess Whitwam  Is a corresponding author
  4. Sebastian Jojoa-Cruz
  5. Stuart M Cahalan  Is a corresponding author
  6. Seyed Ali Reza Mousavi
  7. Andrew B Ward  Is a corresponding author
  8. Ardem Patapoutian  Is a corresponding author
  1. The Scripps Research Institute, United States
  2. The Scripps Research Institute, Howard Hughes Medical Institute, United States
https://doi.org/10.7554/eLife.41844
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Cite this article
  1. Swetha E Murthy
  2. Adrienne E Dubin
  3. Tess Whitwam
  4. Sebastian Jojoa-Cruz
  5. Stuart M Cahalan
  6. Seyed Ali Reza Mousavi
  7. Andrew B Ward
  8. Ardem Patapoutian
(2018)
OSCA/TMEM63 are an Evolutionarily Conserved Family of Mechanically Activated Ion Channels
eLife 7:e41844.
https://doi.org/10.7554/eLife.41844
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4 figures, 3 tables and 1 additional file

Figures

Figure 1 with 3 supplements
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OSCA1.1 and 1.2 induce MA currents in HEK-P1KO cells.

(A) Representative traces of MA whole-cell currents (−80 mV) from OSCA1.1- and OSCA1.2-expressing cells. The corresponding probe displacement trace is illustrated above the current trace. (B) Left, …

https://doi.org/10.7554/eLife.41844.002
Figure 1—figure supplement 1
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Hyperosmolarity-evoked currents from OSCA1.1- and OSCA1.2-transfected HEK-P1KO cells.

(A) Maximal current (inward current) response recorded at −100 mV from cells expressing OSCA1.1 or OSCA1.2 in iso-osmotic (300 mmol.kg-1) or hyperosmotic solutions (620 mmol.kg−1). In the whole-cell …

https://doi.org/10.7554/eLife.41844.003
Figure 1—figure supplement 2
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Subconductance states in OSCA1.1- and OSCA1.2-dependent stretch-activated single-channel traces.

Representative traces from two different cells for OSCA1.1 (A) and OSCA1.2 (B). Channel openings are upward deflections, and currents were recorded at −80 mV but are illustrated as upward currents. …

https://doi.org/10.7554/eLife.41844.004
Figure 1—figure supplement 3
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Gadolinium block of OSCA1.1- and OSCA1.2-dependent MA currents.

(A and C) Representative trace of stretch-activated currents at −80 mV in the cell-attached patch clamp mode. Currents were evoked by −80 mmHg of pressure in the presence of physiological pipette …

https://doi.org/10.7554/eLife.41844.005
Figure 2 with 1 supplement
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Distinct subclasses of OSCA family members induce MA currents in HEK-P1KO cells.

(A) Phylogenetic tree describing sequence relationship between the 15 OSCA family members. Protein sequences were aligned using MegAlign Pro and tree was generated using DrawTree. Genes in orange …

https://doi.org/10.7554/eLife.41844.007
Figure 2—figure supplement 1
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Membrane indentation-induced MA currents in HEK-P1KO cells transfected with different genes from the OSCA family.

MA whole-cell maximal inward currents from individual cells transfected with the indicated gene. The last current response before losing the cell is reported. Currents were elicited at −80 mV …

https://doi.org/10.7554/eLife.41844.008
Figure 3 with 1 supplement
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Reconstituted OSCA1.2 in liposomes form MA ion channels.

(A) Illustration to depict that patches were pulled from liposomes reconstituted with purified MscL or OSCA1.2 proteins. Channels in patch were activated by negative pipette pressure. Brightfield …

https://doi.org/10.7554/eLife.41844.010
Figure 3—figure supplement 1
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Purified OSCA1.2 induces stretch-activated single-channel currents with subconductance states when reconstituted in liposomes.

(A) SDS-PAGE showing purification of EcMscL and OSCA1.2. Samples are taken after eluting protein from resin and before concentrating it for further steps (proteoliposome reconstitution for EcMscL or …

https://doi.org/10.7554/eLife.41844.011
Figure 4 with 3 supplements
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OSCA orthologues in flies and in mammals induce MA currents in HEK-P1KO cells.

(A) Phylogenetic tree illustrating the relationship between OSCA proteins across Arabidopsis thaliana (teal clade), Drosophila melanogaster (purple clade), and Mus musculus and Homo sapiens (orange …

https://doi.org/10.7554/eLife.41844.012
Figure 4—figure supplement 1
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Membrane indentation-induced MA currents in HEK-P1KO cells transfected with TMEM63 homologues.

MA whole-cell maximal inward currents from individual cells transfected with the indicated gene. The last current response before losing the cell is reported. Currents were elicited at −80 mV …

https://doi.org/10.7554/eLife.41844.013
Figure 4—figure supplement 2
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Ion selectivity and gadolinium block of MmTMEM63A-induced MA currents.

(A) I-V relation curves for stretch-activated currents recorded in the cell-attached patch clamp mode from cells expressing MmTMEM63A in the indicated ion solution (Control: N = 11, NMDG-Cl: N = 10, …

https://doi.org/10.7554/eLife.41844.014
Figure 4—figure supplement 3
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Surface expression of MmTMEM63A.

Representative images of Myc labeling in MmTMEM63A-IRESmCherry transfected HEK-P1KO cells. Myc tag was inserted at the N terminal of the protein which was predicted to be on the extracellular side …

https://doi.org/10.7554/eLife.41844.015

Tables

Table 1
Biophysical properties of OSCA and TMEM63 proteins.
https://doi.org/10.7554/eLife.41844.006
GeneWhole-cell/PokeCell-attached/Stretch
Imax
(pA)		Inactivation tau
(ms)		Threshold
(µm)		Imax
(pA)		Activation tau
(ms)		Inactivation tau
(ms)		P50
(-mmHg)		Channel conductance (pS)
Mock4.2 ± 0.5
(10)		--2.32 ± 0.3
(17)		----
Mm
PIEZO1		3045 ± 875
(5)		18.7 ± 1.7
(5)		4.0 ± 0.6
(5)		122 ± 40
(5)		9.0 ± 1.0
(5)		28.8 ± 2.0
(5)		24.0 ± 3.6
(4)		27.3 ± 0.3 (4)*
OSCA1.12271 ± 918
(16)		10.0 ± 1.3
(8)		8.6 ± 0.9
(9)		563 ± 68
(11)		6.7 ± 1.0
(11)		24.0 ± 3.0
(8)		58.5 ± 2.4
(6)		184.4 ± 4.4
(5)
OSCA1.24039 ± 1046
(12)		10.4 ± 1.7
(9)		6.3 ± 0.7
(10)		576 ± 65
(14)		5.5 ± 0.5
(14)		24.6 ± 4.7
(9)		54.5 ± 2.2 (10)121.8 ± 3.4
(4)
OSCA1.87.1 ± 0.7
(11)		--347 ± 56
(7)		14.0 ± 2.4
(7)		67.0 ± 13.0
(6)		79.3 ± 9.9
(6)		46.6 ± 2.8
(4)
OSCA2.354.34 ± 48.9
(11)		--31.3 ± 11
(7)		18.8 ± 3.2
(6)		110.0 ± 70.0
(6)		59.7 ± 3.8
(7)		n.d.
OSCA3.19.7 ± 1.4
(7)		--306 ± 47
(12)		5.6 ± 1.4
(8)		18.5 ± 2.5
(11)		44.5 ± 3.2
(5)		24.9 ± 3.4
(4)
OSCA4.113.3 ± 2.0
(6)		--1.2 ± 0.05
(6)		----
Dm
TMEM63		9.8 ± 2.0
(11)		--14.2 ± 4.5
(9)		22.6 ± 4.3
(5)		154 ± 34
(5)		90 ± 2
(3)		n.d.
Mm
TMEM63A		2.0 ± 1.2
(13)		--18.75 ± 3.0
(23)		126 ± 19
(8)		323 ± 30
(9)		60 ± 5.4
(6)		n.d.
Mm
TMEM63B		3.8 ± 1.8
(7)		--17.97 ± 3.9
(12)		245 ± 38
(7)		323 ± 30
(7)		66 ± 7.8
(4)		n.d.
Mm
TMEM63C		4.5 ± 0.9
(8)		--4.73 ± 0.8
(7)		----
Hs
TMEM63A		4.2 ± 0.7
(6)		--12.4 ± 4.4
(9)		188 ± 18
(5)		427 ± 94
(2)		59.7 ± 3.2
(5)		n.d.

  1. Note: n.d.: not determined. Ns are indicated in parenthesis. Imax value is reported for the last indentation or stretch stimulus before losing the cell. Activation is reported as 10–90% rise of stretch-activated current at saturating stimulus. Inactivation time constant for stretch-activated currents are reported in the range of −60 to −80 mmHg stimulus-pressure. All values are mean ± s.e.m. * values as indicated in Saotome et al., 2018

Table 2
Percent identity in protein sequence among OSCA and TMEM63 family of genes.
https://doi.org/10.7554/eLife.41844.009
GeneOsca
1.1		Osca
1.2		Osca
1.8		Osca
2.3		Osca
3.1		Osca
4.1		Dm
TMEM63		Mm
TMEM63A		Mm
TMEM63B		Mm
TMEM63C		Hs
TMEM63A
OSCA
1.1		10084.815832.7629.0918.8218.0719.1919.3217.5520.11
OSCA
1.2		-10058.3934.4728.9519.4218.4020.0919.5217.7520.43
OSCA
1.8		--10032.2431.7918.0018.3219.2619.0619.1519.46
OSCA
2.3		---10023.8017.301701219.5718.1717.8819.41
OSCA
3.1		----10018.3918.9222.3620.3319.7921.75
OSCA
4.1		-----10018.9721.2721.3019.6821.42
Dm
TMEM63		------10032.5832.7930.7832.59
Mm
TMEM63A		-------10057.1641.2890.05
Mm
TMEM63B		--------10044.4657.58
Mm
TMEM63C		---------10040.73
Hs
TMEM63A		----------100
Key resources table
Reagent type
(species) or
resource		DesignationSource or
reference		IdentifiersAdditional
information
Cell line
(Piezo1
knockout
HEK293T cells)		HEK-P1KO
cells		PMID: 28426961Patapoutian
Lab (Scripps
Research)
Antibody9E11 anti-
Myc antibody
(primary)		Santa Cruz
Biotechnology		Cat# sc-47694,
RRID:AB_627266		1:50
AntibodyAlexa Fluor
488 (secondary)		InvitrogenCat# A-21121,
RRID:AB_141514		1:200
Transfected
construct
(Arabidopsis
thaliana)		OSCA1.1this paperNM_178966.1,
At4g04340		Human codon
optimized, Gene
synthesized from
Genewiz
Transfected
construct
(Arabidopsis
thaliana)		OSCA1.2this paperNM_118333.5,
At4g22120		Human codon
optimized, Gene
synthesized from
Genewiz
Transfected
construct
(Arabidopsis
thaliana)		OSCA1.8this paperNM_102943.6,
At1g32090		Human
codon optimized,
Gene synthesized
from Genewiz
Transfected
construct
(Arabidopsis
thaliana)		OSCA2.3this paperNM_110975.5,
At3g01100		Human codon
optimized, Gene
synthesized from
Genewiz
Transfected
construct
(Arabidopsis
thaliana)		OSCA3.1this paperNM_102773.3,
At1g30360		Human codon
optimized, Gene
synthesized from
Genewiz
Transfected
construct
(Arabidopsis
thaliana)		OSCA4.1this paperNM_119753.3,
At4g35870		Human codon
optimized, Gene
synthesized from
Genewiz
Transfected
construct
(Drosophila
melanogaster)		DmTMEM63this paperDmel_CG11210Human codon
optimized, Gene
synthesized from
Genewiz
Transfected
construct (Mus
musculus)		MmTMEM63AORIGENENM_144794,
Cat No. MR210748
Transfected
construct (Mus
musculus)		MmTMEM63BORIGENENM_198167,
Cat No. MR221527
Transfected
construct (Mus
musculus)		MmTMEM63CORIGENENM_172583,
Cat No. MR210738
Transfected
construct (Homo
sapiens)		HsTMEM63AORIGENENM_014698,
Cat No. RC206992

Additional files

Transparent reporting form
https://doi.org/10.7554/eLife.41844.016

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