Difference between revisions of "Part:BBa K1965002"

 
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<partinfo>BBa_K1965002 short</partinfo>
 
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<p>This part contains the coding sequence of P3:FAStm:TRPC1, a designed mechanosensitive channel. The transient receptor potential channel 1 TRPC1 (<a href="http://2016.igem.org/Team:Slovenia/Mechanosensing/Mechanosensitive_channels">Read more</a>) is a human non-specific cation channel <sup>[1]</sup>, which was we fused to an additional FAS transmembrane domain in order to improve plasma membrane localization. Addition of the FAS transmembrane domain was previously used for similar purposes <sup>[2]</sup>. The addition of the FAS transmembrane domain to the N-terminal of TRPC1 would affect the localization of the N-terminus, changing it from cytoplasmic in native protein to extracellular in the presence of FAS transmembrane domain. This modification also presents an additional advantage, since our construct P3:FAStm:TRPC1 (where P3 stands for coiled coil) would be able to interact with proteins, fused with the P3 coiled coil peptide pair AP4 from outside the cell.</p>
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<p>We used theP3:FAStm:TRPC1 mechanosensitive channel as a source of Ca2+ influx when stimulated with ultrasound <ref>1</ref>.</p>
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        <img class="ui medium image" src="https://static.igem.org/mediawiki/2016/f/fd/T--Slovenia--S.3.1.2.png">
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        <figcaption><b> Schematic presentation of MscS channel function in our system.</b><br/> Cells in resting stage with closed P3:FAStm:TRPC1 channels in the plasma membrane (left). Upon ultrasound stimulation P3:FAStm:TRPC1 channels open, leading to Ca2+ influx (right).
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<h3>Characterization</h3>
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<p>Subcellular localization of P3:FAStm:TRPC1 channels in HEK293T cells was inspected. HEK293T cells were transfected with plasmids encoding HA- and Myc-tagged P3:FAStm:TRPC1 channel and protein localization was investigated by confocal microscopy.</p>
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        <img class="ui medium image" src=" https://static.igem.org/mediawiki/2016/1/13/T--Slovenia--3.2.3.png">
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        <figcaption><b> Subcellular localization of fusion protein P3:FAStm:TRPC1. </b><br/>(A) Scheme of ion channel P3:FAStm:TRPC1.
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(B) Ion channel P3:FAStm:TRPC1 is localized on the plasma membrane. Cells were permeabilized (upper) or non-permeabilized (lower) and stained with antibodies against HA and Myc-tag. Localization on plasma membrane is shown with arrows.
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        </figcaption>
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<h3>Ultrasound stimulation</h3>
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<p>After confirming P3:FAStm:TRPC1 localization on the plasma membrane in HEK293 cells, we stimulated the transfected cells with ultrasound to verify and characterize channel activity. Our experimental setup included an in-house built hardware MODUSON connected to unfocused transducer Olympus V318-SU.  To monitor cell response in situ and in real time we used standard ratiometric fluorescent calcium indicators Fura Red, AM and Fluo-4, AM, which can be easily detected with confocal microscopy.. When activated, mechanosensitive channels open, leading to calcium influx, which in turn binds the fluorescent calcium indicators. The indicator conformation changes upon calcium binding, resulting in an increase or a decrease of fluorescence.</p>
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<p>Fusion of the FAS transmembrane domain to TRPC1 did not only improve its membrane localization, but also significantly enhanced its sensitivity to ultrasound stimulation <ref>3</ref> suggesting the importance of membrane localization in the function of mechanosensitive receptors.</p>
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<div>
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    <figure data-ref="3">
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        <img class="ui medium image" src="https://static.igem.org/mediawiki/2016/8/8a/T--Slovenia--3.2.4.png">
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        <figcaption><b> P3:FAStm:TRPC1 channel improves sensitivity of cells for ultrasound </b><br/>(A) Schematic presentation of the stimulation sequence and (B) signal parameters used for stimulation.
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(C, D)Cells expressing the P3:FAStm:TRPC1 channel showed increased sensitivity/responsiveness to ultrasound stimulation in comparison to the cells without exogenous mechanosensitive channel. HEK293 cells either expressing P3:FAStm:TRPC1 or transfected with a mock plasmid were stimulated with ultrasound for 10 s and calcium influx was recorded in real time (D) using confocal microscopy. Changes in fluorescence intensity of calcium indicators Fluo-4, AM (green line) and Fura Red, AM (red line) are shown. The blue line represents the ratio of Fluo-4 and Fura Red intensities, indicating the increase in intracellular free calcium ions after ultrasound stimulation.
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        </figcaption>
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    </figure>
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</div>
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<h3> References </h3>
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<sup>[1]</sup>Xu, Shang-zhong, and David J Beech. 2001. “TrpC1 Is a Membrane-Spanning Subunit of Store-Operated Ca2+ Channels in Native Vascular Smooth Muscle Cells.” Circulation Research 88: 84–87. <br>
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<sup>[2]</sup>Majerle, Andreja, Rok Gaber, Mojca Benčina, and Roman Jerala. 2015. “Function-Based Mutation-Resistant Synthetic Signaling Device Activated by HIV-1 Proteolysis.” ACS Synthetic Biology 4(6): 667–72. <br>
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Revision as of 16:14, 18 October 2016


P3:FAStm:HA:TRPC1:Myc

This part contains the coding sequence of P3:FAStm:TRPC1, a designed mechanosensitive channel. The transient receptor potential channel 1 TRPC1 (Read more) is a human non-specific cation channel [1], which was we fused to an additional FAS transmembrane domain in order to improve plasma membrane localization. Addition of the FAS transmembrane domain was previously used for similar purposes [2]. The addition of the FAS transmembrane domain to the N-terminal of TRPC1 would affect the localization of the N-terminus, changing it from cytoplasmic in native protein to extracellular in the presence of FAS transmembrane domain. This modification also presents an additional advantage, since our construct P3:FAStm:TRPC1 (where P3 stands for coiled coil) would be able to interact with proteins, fused with the P3 coiled coil peptide pair AP4 from outside the cell.

We used theP3:FAStm:TRPC1 mechanosensitive channel as a source of Ca2+ influx when stimulated with ultrasound 1.

Schematic presentation of MscS channel function in our system.
Cells in resting stage with closed P3:FAStm:TRPC1 channels in the plasma membrane (left). Upon ultrasound stimulation P3:FAStm:TRPC1 channels open, leading to Ca2+ influx (right).

Characterization

Subcellular localization of P3:FAStm:TRPC1 channels in HEK293T cells was inspected. HEK293T cells were transfected with plasmids encoding HA- and Myc-tagged P3:FAStm:TRPC1 channel and protein localization was investigated by confocal microscopy.

Subcellular localization of fusion protein P3:FAStm:TRPC1.
(A) Scheme of ion channel P3:FAStm:TRPC1. (B) Ion channel P3:FAStm:TRPC1 is localized on the plasma membrane. Cells were permeabilized (upper) or non-permeabilized (lower) and stained with antibodies against HA and Myc-tag. Localization on plasma membrane is shown with arrows.

Ultrasound stimulation

After confirming P3:FAStm:TRPC1 localization on the plasma membrane in HEK293 cells, we stimulated the transfected cells with ultrasound to verify and characterize channel activity. Our experimental setup included an in-house built hardware MODUSON connected to unfocused transducer Olympus V318-SU. To monitor cell response in situ and in real time we used standard ratiometric fluorescent calcium indicators Fura Red, AM and Fluo-4, AM, which can be easily detected with confocal microscopy.. When activated, mechanosensitive channels open, leading to calcium influx, which in turn binds the fluorescent calcium indicators. The indicator conformation changes upon calcium binding, resulting in an increase or a decrease of fluorescence.

Fusion of the FAS transmembrane domain to TRPC1 did not only improve its membrane localization, but also significantly enhanced its sensitivity to ultrasound stimulation 3 suggesting the importance of membrane localization in the function of mechanosensitive receptors.

P3:FAStm:TRPC1 channel improves sensitivity of cells for ultrasound
(A) Schematic presentation of the stimulation sequence and (B) signal parameters used for stimulation. (C, D)Cells expressing the P3:FAStm:TRPC1 channel showed increased sensitivity/responsiveness to ultrasound stimulation in comparison to the cells without exogenous mechanosensitive channel. HEK293 cells either expressing P3:FAStm:TRPC1 or transfected with a mock plasmid were stimulated with ultrasound for 10 s and calcium influx was recorded in real time (D) using confocal microscopy. Changes in fluorescence intensity of calcium indicators Fluo-4, AM (green line) and Fura Red, AM (red line) are shown. The blue line represents the ratio of Fluo-4 and Fura Red intensities, indicating the increase in intracellular free calcium ions after ultrasound stimulation.

References

[1]Xu, Shang-zhong, and David J Beech. 2001. “TrpC1 Is a Membrane-Spanning Subunit of Store-Operated Ca2+ Channels in Native Vascular Smooth Muscle Cells.” Circulation Research 88: 84–87.
[2]Majerle, Andreja, Rok Gaber, Mojca Benčina, and Roman Jerala. 2015. “Function-Based Mutation-Resistant Synthetic Signaling Device Activated by HIV-1 Proteolysis.” ACS Synthetic Biology 4(6): 667–72.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 655
    Illegal BglII site found at 3099
    Illegal BamHI site found at 649
    Illegal BamHI site found at 850
    Illegal BamHI site found at 1494
    Illegal XhoI site found at 2789
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 73
  • 1000
    COMPATIBLE WITH RFC[1000]