Limits...
A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function.

Kaur G, Pinggera A, Ortner NJ, Lieb A, Sinnegger-Brauns MJ, Yarov-Yarovoy V, Obermair GJ, Flucher BE, Striessnig J - J. Biol. Chem. (2015)

Bottom Line: Neutralization of four arginine residues eliminated plasma membrane binding.Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane.Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca(2+) channel function.

View Article: PubMed Central - PubMed

Affiliation: From the Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.

Show MeSH

Related in: MedlinePlus

Modulation of Cav1.2S and Cav1.2S4E currents by wortmannin/m-3M3FBS or diC8-PIP2 treatment.A, Cav1.2S, perfused with bath solution (control; gray circles; data are shown for seven cells; due to different recording duration, means ± S.E. reflect n = 7 until 410 s, n = 6 until 450 s), 100 μm intracellular diC8-PIP2 (gray triangles; means ± S.E., n = 4), or extracellular 20 μm wortmannin and 50 μm m-3M3FBS (black squares; means ± S.E. for n = 8 until 280 s, n = 3 until 450 s). B, Cav1.2S4E, control (red circles; n = 9 cells until 440 s, n = 8 cells until 450 s), 100 μm intracellular diC8 (red triangles; n = 4 until 420 s, n = 3 until 450 s), or extracellular 20 μm wortmannin and 50 μm m-3M3FBS (white squares; n = 8 until 320 s, n = 6 until 450 s). Statistical analysis was performed using two-way analysis of variance of all data sets followed by Bonferroni post hoc test. Wortmannin/m-3M3FBS treatment significantly enhanced current decrease over time for both constructs (a, p < 0.01 after 100 s and p < 0.001 after 120 s for Cav1.2S; b, p < 0.05 after 140 s and p < 0.001 after 170 s for Cav1.2S4E). The wortmannin/m-3M3FBS response was significantly attenuated in Cav1.2S4Eversus wild type (c, p < 0.05 after 260 s, p < 0.01 or p < 0.001 after 330 s). DiC8-PIP2 treatment had no effect and was also not different between wild type and mutant Cav1.2. Because current underwent some minor initial run-up, time 0 was set when the currents reached a stable maximum (1.5–2 min after start of perfusion) to which current amplitudes were normalized. C, percentage of remaining current of Cav1.2S and Cav1.2S4E in control-, diC8-, or wortmannin/m-3M3FBS (W/FBS)-treated cells after different time points. Data are shown as mean ± S.E. (error bars). Statistical significance at the indicated time points was taken from the statistical analysis described above. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4543666&req=5

Figure 7: Modulation of Cav1.2S and Cav1.2S4E currents by wortmannin/m-3M3FBS or diC8-PIP2 treatment.A, Cav1.2S, perfused with bath solution (control; gray circles; data are shown for seven cells; due to different recording duration, means ± S.E. reflect n = 7 until 410 s, n = 6 until 450 s), 100 μm intracellular diC8-PIP2 (gray triangles; means ± S.E., n = 4), or extracellular 20 μm wortmannin and 50 μm m-3M3FBS (black squares; means ± S.E. for n = 8 until 280 s, n = 3 until 450 s). B, Cav1.2S4E, control (red circles; n = 9 cells until 440 s, n = 8 cells until 450 s), 100 μm intracellular diC8 (red triangles; n = 4 until 420 s, n = 3 until 450 s), or extracellular 20 μm wortmannin and 50 μm m-3M3FBS (white squares; n = 8 until 320 s, n = 6 until 450 s). Statistical analysis was performed using two-way analysis of variance of all data sets followed by Bonferroni post hoc test. Wortmannin/m-3M3FBS treatment significantly enhanced current decrease over time for both constructs (a, p < 0.01 after 100 s and p < 0.001 after 120 s for Cav1.2S; b, p < 0.05 after 140 s and p < 0.001 after 170 s for Cav1.2S4E). The wortmannin/m-3M3FBS response was significantly attenuated in Cav1.2S4Eversus wild type (c, p < 0.05 after 260 s, p < 0.01 or p < 0.001 after 330 s). DiC8-PIP2 treatment had no effect and was also not different between wild type and mutant Cav1.2. Because current underwent some minor initial run-up, time 0 was set when the currents reached a stable maximum (1.5–2 min after start of perfusion) to which current amplitudes were normalized. C, percentage of remaining current of Cav1.2S and Cav1.2S4E in control-, diC8-, or wortmannin/m-3M3FBS (W/FBS)-treated cells after different time points. Data are shown as mean ± S.E. (error bars). Statistical significance at the indicated time points was taken from the statistical analysis described above. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Mentions: We also tested whether the arginine mutations in CaV1.2S4E (versus CaV1.2S as wild-type control) affect the time-dependent decline of whole-cell ICa and the modulation by added PIP2 (by intracellular application of a 100 μm concentration of the water-soluble PIP2 analogue diC8-PIP2, (15, 64)) or phosphoinositide hydrolysis induced by wortmannin (20 μm) plus m-3M3FBS (50 μm) as in live cell imaging experiments (Fig. 5A). Perfusion of cells with control solution induced a slow decrease in activity with time (“run-down”), as expected for Cav1.2 channels (65). ICa decline during perfusion with control solution was similar in wild-type and the mutant channel and was also not affected by intracellular application of diC8-PIP2 (Fig. 7). However, extracellular perfusion with wortmannin/m-3M3FBS significantly inhibited ICa of wild-type and Cav1.24E channels, but this current inhibition was significantly attenuated in the mutant channel (Fig. 7). This indicates that membrane association of the distal I-II linker through its positive charges not only stabilizes a more reluctant channel state (Fig. 6) but also weakens inhibition of channel activity by phosphoinositide depletion.


A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function.

Kaur G, Pinggera A, Ortner NJ, Lieb A, Sinnegger-Brauns MJ, Yarov-Yarovoy V, Obermair GJ, Flucher BE, Striessnig J - J. Biol. Chem. (2015)

Modulation of Cav1.2S and Cav1.2S4E currents by wortmannin/m-3M3FBS or diC8-PIP2 treatment.A, Cav1.2S, perfused with bath solution (control; gray circles; data are shown for seven cells; due to different recording duration, means ± S.E. reflect n = 7 until 410 s, n = 6 until 450 s), 100 μm intracellular diC8-PIP2 (gray triangles; means ± S.E., n = 4), or extracellular 20 μm wortmannin and 50 μm m-3M3FBS (black squares; means ± S.E. for n = 8 until 280 s, n = 3 until 450 s). B, Cav1.2S4E, control (red circles; n = 9 cells until 440 s, n = 8 cells until 450 s), 100 μm intracellular diC8 (red triangles; n = 4 until 420 s, n = 3 until 450 s), or extracellular 20 μm wortmannin and 50 μm m-3M3FBS (white squares; n = 8 until 320 s, n = 6 until 450 s). Statistical analysis was performed using two-way analysis of variance of all data sets followed by Bonferroni post hoc test. Wortmannin/m-3M3FBS treatment significantly enhanced current decrease over time for both constructs (a, p < 0.01 after 100 s and p < 0.001 after 120 s for Cav1.2S; b, p < 0.05 after 140 s and p < 0.001 after 170 s for Cav1.2S4E). The wortmannin/m-3M3FBS response was significantly attenuated in Cav1.2S4Eversus wild type (c, p < 0.05 after 260 s, p < 0.01 or p < 0.001 after 330 s). DiC8-PIP2 treatment had no effect and was also not different between wild type and mutant Cav1.2. Because current underwent some minor initial run-up, time 0 was set when the currents reached a stable maximum (1.5–2 min after start of perfusion) to which current amplitudes were normalized. C, percentage of remaining current of Cav1.2S and Cav1.2S4E in control-, diC8-, or wortmannin/m-3M3FBS (W/FBS)-treated cells after different time points. Data are shown as mean ± S.E. (error bars). Statistical significance at the indicated time points was taken from the statistical analysis described above. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543666&req=5

Figure 7: Modulation of Cav1.2S and Cav1.2S4E currents by wortmannin/m-3M3FBS or diC8-PIP2 treatment.A, Cav1.2S, perfused with bath solution (control; gray circles; data are shown for seven cells; due to different recording duration, means ± S.E. reflect n = 7 until 410 s, n = 6 until 450 s), 100 μm intracellular diC8-PIP2 (gray triangles; means ± S.E., n = 4), or extracellular 20 μm wortmannin and 50 μm m-3M3FBS (black squares; means ± S.E. for n = 8 until 280 s, n = 3 until 450 s). B, Cav1.2S4E, control (red circles; n = 9 cells until 440 s, n = 8 cells until 450 s), 100 μm intracellular diC8 (red triangles; n = 4 until 420 s, n = 3 until 450 s), or extracellular 20 μm wortmannin and 50 μm m-3M3FBS (white squares; n = 8 until 320 s, n = 6 until 450 s). Statistical analysis was performed using two-way analysis of variance of all data sets followed by Bonferroni post hoc test. Wortmannin/m-3M3FBS treatment significantly enhanced current decrease over time for both constructs (a, p < 0.01 after 100 s and p < 0.001 after 120 s for Cav1.2S; b, p < 0.05 after 140 s and p < 0.001 after 170 s for Cav1.2S4E). The wortmannin/m-3M3FBS response was significantly attenuated in Cav1.2S4Eversus wild type (c, p < 0.05 after 260 s, p < 0.01 or p < 0.001 after 330 s). DiC8-PIP2 treatment had no effect and was also not different between wild type and mutant Cav1.2. Because current underwent some minor initial run-up, time 0 was set when the currents reached a stable maximum (1.5–2 min after start of perfusion) to which current amplitudes were normalized. C, percentage of remaining current of Cav1.2S and Cav1.2S4E in control-, diC8-, or wortmannin/m-3M3FBS (W/FBS)-treated cells after different time points. Data are shown as mean ± S.E. (error bars). Statistical significance at the indicated time points was taken from the statistical analysis described above. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Mentions: We also tested whether the arginine mutations in CaV1.2S4E (versus CaV1.2S as wild-type control) affect the time-dependent decline of whole-cell ICa and the modulation by added PIP2 (by intracellular application of a 100 μm concentration of the water-soluble PIP2 analogue diC8-PIP2, (15, 64)) or phosphoinositide hydrolysis induced by wortmannin (20 μm) plus m-3M3FBS (50 μm) as in live cell imaging experiments (Fig. 5A). Perfusion of cells with control solution induced a slow decrease in activity with time (“run-down”), as expected for Cav1.2 channels (65). ICa decline during perfusion with control solution was similar in wild-type and the mutant channel and was also not affected by intracellular application of diC8-PIP2 (Fig. 7). However, extracellular perfusion with wortmannin/m-3M3FBS significantly inhibited ICa of wild-type and Cav1.24E channels, but this current inhibition was significantly attenuated in the mutant channel (Fig. 7). This indicates that membrane association of the distal I-II linker through its positive charges not only stabilizes a more reluctant channel state (Fig. 6) but also weakens inhibition of channel activity by phosphoinositide depletion.

Bottom Line: Neutralization of four arginine residues eliminated plasma membrane binding.Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane.Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca(2+) channel function.

View Article: PubMed Central - PubMed

Affiliation: From the Institute of Pharmacy, Department of Pharmacology and Toxicology, and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria.

Show MeSH
Related in: MedlinePlus