Limits...
Ca(2+) Channel Re-localization to Plasma-Membrane Microdomains Strengthens Activation of Ca(2+)-Dependent Nuclear Gene Expression.

Samanta K, Kar P, Mirams GR, Parekh AB - Cell Rep (2015)

Bottom Line: In polarized cells or cells with complex geometry, clustering of plasma-membrane (PM) ion channels is an effective mechanism for eliciting spatially restricted signals.For similar levels of channel activity, we find that channel confinement is considerably more effective in stimulating gene expression.Our results identify a long-range signaling advantage to the tight evolutionary conservation of channel clustering and reveal that CRAC channel aggregation increases the strength, fidelity, and reliability of the general process of excitation-transcription coupling.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, Oxford OX1 3PT, UK.

No MeSH data available.


Related in: MedlinePlus

The Mutant V102C-Orai1 Channel Is Constitutively Active and Does Not Form Puncta Characteristic of Store-Operated Orai1 Channels in RBL-1 Cells(A) Western blot compares the amount of endogenous Orai1 protein with levels after expression of V102C-Orai1. The histogram summarizes data from two independent gels.(B) Cytoplasmic Ca2+ measurements compare Ca2+ entry evoked by V102C channels with that induced by thapsigargin (2 μM). Cells expressing V102C-Orai1 channels were initially maintained in external solution containing 2 mM Ca2+ and then perfused with Ca2+-free solution for ∼5 min before external Ca2+ was readmitted. By contrast, thapsigargin-evoked responses in mock-transfected cells were obtained in Ca2+-free solution, and external Ca2+ was readmitted ∼7 min later. The mock recording shows a cell exposed simply to Ca2+-free solution for 7 min before external Ca2+ was readmitted to obtain the basal Ca2+ entry rate in the absence of store depletion.(C) Aggregate data for the various conditions are compared. Each bar is the average of between 24 and 38 cells. Low Na+ refers to external solution containing 10 mM Na+, replaced with Tris+. For all bars, cells were exposed to Ca2+-free solution for 7 min before external Ca2+ was readmitted.(D) Confocal microscopy images compare the distribution of V102C-Orai1-cherry for the conditions shown. STIM1 refers to transfection with STIM1-YFP plasmid.(E) Co-immunoprecipitation studies show that after pull-down of V102C-Orai1-YFP, Syk was detected in the immunoblots, and this association is unaffected by knockdown of STIM1.Error bars represent SEM.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4521080&req=5

fig2: The Mutant V102C-Orai1 Channel Is Constitutively Active and Does Not Form Puncta Characteristic of Store-Operated Orai1 Channels in RBL-1 Cells(A) Western blot compares the amount of endogenous Orai1 protein with levels after expression of V102C-Orai1. The histogram summarizes data from two independent gels.(B) Cytoplasmic Ca2+ measurements compare Ca2+ entry evoked by V102C channels with that induced by thapsigargin (2 μM). Cells expressing V102C-Orai1 channels were initially maintained in external solution containing 2 mM Ca2+ and then perfused with Ca2+-free solution for ∼5 min before external Ca2+ was readmitted. By contrast, thapsigargin-evoked responses in mock-transfected cells were obtained in Ca2+-free solution, and external Ca2+ was readmitted ∼7 min later. The mock recording shows a cell exposed simply to Ca2+-free solution for 7 min before external Ca2+ was readmitted to obtain the basal Ca2+ entry rate in the absence of store depletion.(C) Aggregate data for the various conditions are compared. Each bar is the average of between 24 and 38 cells. Low Na+ refers to external solution containing 10 mM Na+, replaced with Tris+. For all bars, cells were exposed to Ca2+-free solution for 7 min before external Ca2+ was readmitted.(D) Confocal microscopy images compare the distribution of V102C-Orai1-cherry for the conditions shown. STIM1 refers to transfection with STIM1-YFP plasmid.(E) Co-immunoprecipitation studies show that after pull-down of V102C-Orai1-YFP, Syk was detected in the immunoblots, and this association is unaffected by knockdown of STIM1.Error bars represent SEM.

Mentions: Expression of V102C-Orai1 (untagged) in RBL-1 cells led to an approximate doubling of Orai1 levels (Figure 2A), indicating that the recombinant protein was expressed at a similar level to the endogenous channels. We measured constitutive Ca2+ entry following V102C-Orai1 expression by first briefly removing external Ca2+ and then measuring the rate of rise of the cytoplasmic Ca2+ signal that occurred when external Ca2+ was readmitted (Figure 2B). Compared with non-stimulated, mock-transfected cells, where very little Ca2+ entry occurred after 5–7 min exposure to Ca2+–free solution, prominent Ca2+ influx was observed in cells expressing V102C-Orai1 (Figures 2B and 2C). The rate of Ca2+ entry for the mutant was slightly (∼30%) but significantly slower than that seen after challenge with a maximally effective concentration of thapsigargin in mock-transfected cells (dotted line in Figure 2B; Figure 2C). Knockdown of STIM1 did not alter the rate of Ca2+ influx through V102C-Orai1 channels (Figure 2C), consistent with activity independent of the ER Ca2+ sensor. In resting cells, V102C-Orai1-cherry (Figure 2D) was uniformly distributed in the PM with no evidence for the presence of punctate-like fluorescent structures or co-localization with STIM1-YFP (Figure 2D). Neither perfusion in Ca2+-free solution for up to 7 min nor subsequent readmission of external Ca2+ for 10–20 min altered the distribution of either STIM1 or V102C-Orai1 proteins (Figure 2D). V102C-Orai1 also retained the ability to interact with Syk. Pull-down of V102C-Orai1-YFP with an anti-GFP antibody revealed the presence of Syk in resting cells (Figure 2E), and knockdown of STIM1 did not affect this association (Figure 2E).


Ca(2+) Channel Re-localization to Plasma-Membrane Microdomains Strengthens Activation of Ca(2+)-Dependent Nuclear Gene Expression.

Samanta K, Kar P, Mirams GR, Parekh AB - Cell Rep (2015)

The Mutant V102C-Orai1 Channel Is Constitutively Active and Does Not Form Puncta Characteristic of Store-Operated Orai1 Channels in RBL-1 Cells(A) Western blot compares the amount of endogenous Orai1 protein with levels after expression of V102C-Orai1. The histogram summarizes data from two independent gels.(B) Cytoplasmic Ca2+ measurements compare Ca2+ entry evoked by V102C channels with that induced by thapsigargin (2 μM). Cells expressing V102C-Orai1 channels were initially maintained in external solution containing 2 mM Ca2+ and then perfused with Ca2+-free solution for ∼5 min before external Ca2+ was readmitted. By contrast, thapsigargin-evoked responses in mock-transfected cells were obtained in Ca2+-free solution, and external Ca2+ was readmitted ∼7 min later. The mock recording shows a cell exposed simply to Ca2+-free solution for 7 min before external Ca2+ was readmitted to obtain the basal Ca2+ entry rate in the absence of store depletion.(C) Aggregate data for the various conditions are compared. Each bar is the average of between 24 and 38 cells. Low Na+ refers to external solution containing 10 mM Na+, replaced with Tris+. For all bars, cells were exposed to Ca2+-free solution for 7 min before external Ca2+ was readmitted.(D) Confocal microscopy images compare the distribution of V102C-Orai1-cherry for the conditions shown. STIM1 refers to transfection with STIM1-YFP plasmid.(E) Co-immunoprecipitation studies show that after pull-down of V102C-Orai1-YFP, Syk was detected in the immunoblots, and this association is unaffected by knockdown of STIM1.Error bars represent SEM.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4521080&req=5

fig2: The Mutant V102C-Orai1 Channel Is Constitutively Active and Does Not Form Puncta Characteristic of Store-Operated Orai1 Channels in RBL-1 Cells(A) Western blot compares the amount of endogenous Orai1 protein with levels after expression of V102C-Orai1. The histogram summarizes data from two independent gels.(B) Cytoplasmic Ca2+ measurements compare Ca2+ entry evoked by V102C channels with that induced by thapsigargin (2 μM). Cells expressing V102C-Orai1 channels were initially maintained in external solution containing 2 mM Ca2+ and then perfused with Ca2+-free solution for ∼5 min before external Ca2+ was readmitted. By contrast, thapsigargin-evoked responses in mock-transfected cells were obtained in Ca2+-free solution, and external Ca2+ was readmitted ∼7 min later. The mock recording shows a cell exposed simply to Ca2+-free solution for 7 min before external Ca2+ was readmitted to obtain the basal Ca2+ entry rate in the absence of store depletion.(C) Aggregate data for the various conditions are compared. Each bar is the average of between 24 and 38 cells. Low Na+ refers to external solution containing 10 mM Na+, replaced with Tris+. For all bars, cells were exposed to Ca2+-free solution for 7 min before external Ca2+ was readmitted.(D) Confocal microscopy images compare the distribution of V102C-Orai1-cherry for the conditions shown. STIM1 refers to transfection with STIM1-YFP plasmid.(E) Co-immunoprecipitation studies show that after pull-down of V102C-Orai1-YFP, Syk was detected in the immunoblots, and this association is unaffected by knockdown of STIM1.Error bars represent SEM.
Mentions: Expression of V102C-Orai1 (untagged) in RBL-1 cells led to an approximate doubling of Orai1 levels (Figure 2A), indicating that the recombinant protein was expressed at a similar level to the endogenous channels. We measured constitutive Ca2+ entry following V102C-Orai1 expression by first briefly removing external Ca2+ and then measuring the rate of rise of the cytoplasmic Ca2+ signal that occurred when external Ca2+ was readmitted (Figure 2B). Compared with non-stimulated, mock-transfected cells, where very little Ca2+ entry occurred after 5–7 min exposure to Ca2+–free solution, prominent Ca2+ influx was observed in cells expressing V102C-Orai1 (Figures 2B and 2C). The rate of Ca2+ entry for the mutant was slightly (∼30%) but significantly slower than that seen after challenge with a maximally effective concentration of thapsigargin in mock-transfected cells (dotted line in Figure 2B; Figure 2C). Knockdown of STIM1 did not alter the rate of Ca2+ influx through V102C-Orai1 channels (Figure 2C), consistent with activity independent of the ER Ca2+ sensor. In resting cells, V102C-Orai1-cherry (Figure 2D) was uniformly distributed in the PM with no evidence for the presence of punctate-like fluorescent structures or co-localization with STIM1-YFP (Figure 2D). Neither perfusion in Ca2+-free solution for up to 7 min nor subsequent readmission of external Ca2+ for 10–20 min altered the distribution of either STIM1 or V102C-Orai1 proteins (Figure 2D). V102C-Orai1 also retained the ability to interact with Syk. Pull-down of V102C-Orai1-YFP with an anti-GFP antibody revealed the presence of Syk in resting cells (Figure 2E), and knockdown of STIM1 did not affect this association (Figure 2E).

Bottom Line: In polarized cells or cells with complex geometry, clustering of plasma-membrane (PM) ion channels is an effective mechanism for eliciting spatially restricted signals.For similar levels of channel activity, we find that channel confinement is considerably more effective in stimulating gene expression.Our results identify a long-range signaling advantage to the tight evolutionary conservation of channel clustering and reveal that CRAC channel aggregation increases the strength, fidelity, and reliability of the general process of excitation-transcription coupling.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, Oxford OX1 3PT, UK.

No MeSH data available.


Related in: MedlinePlus