Results
Strategy for probing effects of multiple CaM in tuning CaV1 CDI

Construct | Ca2+ r300 at +10 mV | Ba2+ r300 at +10 mV | CDI300 at +10 mV | p value |
---|---|---|---|---|
α1C + β2A | 0.46 ± 0.07 | 1.08 ± 0.09 | 0.57 ± 0.05 (n = 5) | NA |
α1CΔ1671 + β2A | 0.42 ± 0.04 | 0.81 ± 0.08 | 0.48 ± 0.02 (n = 5) | 0.910 |
α1CΔ1671-CaMWT + β2A | 0.42 ± 0.03 | 0.85 ± 0.07 | 0.49 ± 0.04 (n = 5) | >0.9777; >0.9999 |
α1CΔ1671-CaM1234 + β2A | 0.86 ± 0.05 | 0.91 ± 0.01 | 0.05 ± 0.05 (n = 6) | <0.0001; <0.0001 |
α1C + β2A-CaMWT | 0.35 ± 0.02 | 0.96 ± 0.02 | 0.64 ± 0.02 (n = 8) | 0.9688; 0.1326; 0.2591 |
α1C + β2A-CaM1234 | 0.89 ± 0.05 | 0.98 ± 0.04 | 0.09 ± 0.05 (n = 7) | <0.0001; <0.0001 |
α1CΔ1671-CaMWT + β2A-CaMWT | 0.63 ± 0.03 | 1.00 ± 0.02 | 0.36 ± 0.03 (n = 7) | 0.0271; 0.6832 |
α1CΔ1671-CaMWT + β2A-CaM1234 | 0.47 ± 0.04 | 0.92 ± 0.05 | 0.49 ± 0.05 (n = 8) | 0.9095; >0.9999 |
α1CΔ1671-CaM1234 + β2A-CaMWT | 0.80 ± 0.03 | 0.86 ± 0.03 | 0.063 ± 0.04 (n = 6) | <0.0001; <0.0001 |
α1CΔ1671-CaM1234 + β2A + CaM | 0.86 ± 0.04 | 0.89 ± 0.02 | 0.003 ± 0.03 (n = 5) | <0.0001; <0.0001 |
α1C + β2A-CaM1234 + CaM | 0.79 ± 0.07 | 0.90 ± 0.07 | 0.13 ± 0.03 (n = 5) | <0.0001; <0.0001 |
CaV1 preferentially responds to CaM tethered to the channel carboxyl terminus


Construct | Ca22+ r300 | Ba2+ r300 | CDI300 at +10 mV (mean ± S.E.) | p value |
---|---|---|---|---|
α1D + β2A | 0.23 ± 0.02 | 0.97 ± 0.01 | 0.765 ± 0.025 (n = 8) | NA |
α1D + β2A-CaMWT | 0.17 ± 0.06 | 0.94 ± 0.03 | 0.820 ± 0.057 (n = 6) | 0.9231 |
α1D + β2A-CaM1234 | 0.96 ± 0.02 | 1.00 ± 0.01 | 0.033 ± 0.029 (n = 7) | <0.0001 |
α1d-CaMWT + β2A-CaMWT | 0.32 ± 0.03 | 0.98 ± 0.01 | 0.673 ± 0.036 (n = 8) | 0.3858 |
α1d-CaMWT + β2A-CaM1234 | 0.23 ± 0.03 | 0.90 ± 0.03 | 0.746 ± 0.025 (n = 6) | 0.9996 |
α1d-CaM1234 + β2A-CaMWT | 0.97 ± 0.09 | 0.99 ± 0.01 | 0.025 ± 0.084 (n = 5) | <0.0001 |
α1d-CaM1234 + β2A + CaM | 0.91 ± 0.05 | 0.95 ± 0.05 | 0.041 ± 0.047 (n = 5) | <0.0001 |
α1D + β2A-CaM1234 + CaM | 0.83 ± 0.05 | 0.86 ± 0.06 | 0.035 ± 0.025 (n = 5) | <0.0001 |
Functional CaM stoichiometry for CaV1 is limited by the number of CaV IQ domains

Construct | Ca22+ r300 | Ba2+ r300 | CDI300 at +10 mV (mean ± S.E.) | p value |
---|---|---|---|---|
α1D/2×IQ-CaMWT + β2A | 0.13 ± 0.02 | 0.81 ± 0.07 | 0.836 ± 0.020 (n = 5) | 0.3953 |
α1D/2×IQ-CaM1234 + β2A | 0.89 ± 0.04 | 1.01 ± 0.04 | 0.124 ± 0.017 (n = 6) | <0.0001 |
α1D/2×IQ-CaMWT + β2A-CaMWT | 0.26 ± 0.01 | 0.97 ± 0.03 | 0.724 ± 0.017 (n = 6) | 0.7614 |
α1D/2×IQ-CaMWT + β2A-CaM1234 | 0.60 ± 0.05 | 0.94 ± 0.03 | 0.354 ± 0.070 (n = 5) | <0.0001 |
α1D/2×IQ-CaM1234 + β2A-CaMWT | 0.48 ± 0.05 | 0.98 ± 0.01 | 0.478 ± 0.061 (n = 5) | <0.0001 |
α1D/2×IQ-CaM1234 + β2A-CaM1234 | 0.99 ± 0.01 | 1.00 ± 0.01 | 0.011 ± 0.006 (n = 5) | <0.0001 |

Distinct modes of CaV2.1 regulation are preferentially evoked by carboxyl-terminally linked CaM




Discussion
Experimental procedures
Molecular biology
Cell culture and transfection of HEK293 cells
Whole-cell electrophysiology recordings
Data availability
Acknowledgments
Supplementary Material
References
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Author contributions—N. C., P. S. Y., and M. B.-J. conceptualization; N. C. and J. D. data curation; N. C., J. D., and M. B.-J. formal analysis; N. C. and M. B.-J. investigation; N. C. and M. B.-J. methodology; N. C. and M. B.-J. writing-original draft; N. C., J. D., P. S. Y., and M. B.-J. writing-review and editing; P. S. Y. and M. B.-J. resources; M. B.-J. supervision; M. B.-J. funding acquisition.
Funding and additional information—This work was supported by NINDS, National Institutes of Health, Grant NS110672 (to M. B. J.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.
Abbreviations—The abbreviations used are: CaM
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