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. 2018 Feb 19;8:3263. doi: 10.1038/s41598-018-21586-5

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Whole-cell currents from HEK293t cells expressing Aplysia RFP-Ca_V2α1 wildtype or RFP-Ca_V2α1 Y1501F, and Ca_Vβ and Ca_Vα2δ. (A) Representative traces from activation (left column) and steady-state inactivation (right column) voltage-clamp experiments. Top row are the command voltage steps overlaid, followed by the currents overlaid from representative cells without RFP, and RFP expressing cells transfected with either RFP-Ca_V2α1 wt or RFP-Ca_V2α1 Y1501F (along with Ca_Vβ and Ca_Vα2δ). The grey trace is the voltage step to −40 mV in the activation protocol and to 0 mV in the steady-state inactivation protocol, scale bars are 20 pA, 50/200 ms. (B) Activation curves generated from the protocol displayed in A for RFP-Ca_V2α1 (WT) and RFP-Ca_V2α1 Y1501F (Y/F) normalized to the peak current for each group. Data included only for cells with a larger peak inward current than outward current at the end of the step, to remove trials with larger residual potassium currents (n = 4,4). One trial from the Y1501F group was removed from the averaged data as an outlier at >10 pA/pF. (C) Comparison of mean current density between cells expressing RFP-Ca_V2α1 and RFP-Ca_V2α1 Y1501F (t-test P = 0.8632, n = 10,10). (D) Comparison of mean inactivation tau of peak currents between cells expressing RFP-Ca_V2α1 and RFP-Ca_V2α1 Y1501F (t-test, P = 0.1990, n = 10, 10). (E) Steady-state inactivation curves generated with 1 s conditioning pulses followed by a step to the test potential of + 20 mV comparing cells expressing either RFP-Ca_V2α1 (WT) or RFP-Ca_V2α1 Y1501F (Y/F) (n = 7,7).