Supporting Information

Supporting Information Ravindran et al. 10.1073/pnas.0711624105 SI Materials and Methods Assay of Endogenous Cav␤ Subunits in COS1 Cells. COS-1 cells (⬇106

cells per dish) were transfected with plasmids coding for ␣1C, ␣2␦, and Venus or ECFPN-CaM under standard conditions used in electrophysiological experiments; 72 h after transfection, RNA was isolated with an RNeasy mini kit (Qiagen), and 4 ␮g of the RNA was subjected to reverse transcription (RT) reaction (80 ␮l) with an oligo(dT)12–18 primer by using a Omniscript RT kit (Qiagen). PCR amplification with 2 ␮l of RT mixture and sense (5⬘-GTGGCATTTGCSGTKMGGAC-3⬘) plus antisense (5⬘YCKCCCKATCCACCAGTCATT-3⬘) primers designed to recognize all monkey Cav␤ subunits showed the presence of ␤1 and ␤3 mRNA as confirmed by sequencing analysis. Only traces of ␤2 were observed. For the quantitative real-time PCR assay (qPCR), the following sense and antisense monkey specific

primers, respectively, were designed: 5⬘-CTGGCTAAGCGCTCAGTTCT-3⬘ and 5⬘-AGCATCCAGAGCAACCAACT-3⬘ for ␤1; 5⬘-GCAGAAATCGACAGAGCACA-3⬘ and 5⬘-CGCCCTTCAAATCTGTGTTT-3⬘ for ␤2; 5⬘-ACTAGGCTCCCATTCCAGGT-3⬘ and 5⬘-ATCCCTTCCCTGAGTCTGGT-3⬘ for ␤3. The sense 5⬘-TGACAACAGCCTCAAGATCG-3⬘ and antisense 5⬘-GTCTTCTGGGTGGCAGTGAT-2⬘ primers were used for monkey GAPDH (internal standard). The qPCR was performed with 2 ␮l of the RT product/50 ␮l reaction mixture and a platinumR SYBERR Green qPCR SuperMix-UDG with ROX (Invitrogen), by using a 7300 Real Time PCR System (Applied Biosystems). Agarose gel electrophoresis confirmed that all qPCR products showed single band of an expected size. The mRNA level of each Cav␤ subunit was normalized to that of GAPDH as described (1), and Student’s t test was used for statistical analysis.

1. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-⌬⌬CT method. Methods 25:402– 408.

Ravindran et al. www.pnas.org/cgi/content/short/0711624105

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A

1 ECFPN-CaM

Endogenous CaM ECFPN-CaM

2

kDa

a 50 IB: anti-CaM

b

22

c

50

Venus β-actin

B

IP:

ECFPN-CaM:

IB: anti-LC

36 d

50

anti-FLAG

-

+

IB: anti-β-actin

input

-

+

FLAGN-α1C

250

ECFPN-CaM

50 22

IB: anti-FLAG

IB: anti-CaM

Endogenous CaM Fig. S1. Assessment of CaMex and endogenous CaM in COS1 cells. (A) Expression. COS1 cells were transfected with ␣1C and ␣2␦ plus Venus (lane 1) or ECFPN-CaM (lane 2) as described in Materials and Methods. Shown are Western blot analyses of ECFPN-CaM (a) and endogenous CaM (b) detected on immunoblots (IB) by anti-CaM Ab, and of Venus and ECFPN-CaM detected by anti-LC Ab (c). ␤-actin detected by anti-␤-actin Ab (d) was used as a loading control. (B) Coimmunoprecipitation (IP) with FLAGN-␣1C. COS-1 cells were cotransfected with FLAGN-␣1C, ␣2␦ plus Venus or ECFPN-CaM. Co-IP with FLAG affinity gel was performed. Exogenous ECFPN-CaM was pulled down with FLAGN-␣1C, however endogenous CaM was not detectable in the coimmunoprecipitates of COS1 cells not transfected (left lane) or transfected with ECFPN-CaM (right lane). Molecular mass calibration in kDa is shown at right.


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A

α1C/α2δ/ECFPN-CaMex +DHP

+40 mV 100 pA 200 ms

B

α1C/α2δ/CaMex(wt)

+40 mV 50 pA 200 ms

Normalized ICa

C 0 -0.2 -0.4 -0.6 -0.8 -1.0 -40

0 40 V (mV)

80

Fig. S2. Unlabeled CaM supports gating of Cav␤-deficient Cav1.2 calcium channel similar to that by ECFPN-CaM. EYFPN-␣1C and ␣2␦ were coexpressed in COS1 cells with unlabeled CaM. (A) Inhibition by dihydropyridine Ca2⫹ channel blocker of ICa through the ␣1C/␣2␦/ECFPN-CaMex channel. Shown are superimposed traces of maximal ICa recorded before and after (⫹DHP) 10-min application of 2 ␮M (⫹)PN200 –110. Vt ⫽ ⫹40 mV, Vh ⫽ -90 mV. (B) Representative trace of ICa through the wild-type CaMex-activated ␤-deficient Cav1.2 channel evoked by a test pulse to ⫹40 mV applied from Vh ⫽ -90 mV. (C) Average normalized I-V relationship for ICa through the EYFPN-␣1C/␣2␦/CaMex channel. V0.5 ⫽ 16.7 ⫾ 1.1, kI-V ⫽ -8.3 ⫾ 0.7, Erev ⫽ 110.2 ⫾ 3.3 mV (n ⫽ 6). The calculated parameters are not significantly different from those obtained with ECFPN-CaM (see legend to Fig. 2D) suggesting that properties of the wild-type (endogenous) CaM are similar to those of ECFPN-CaM in the observed Cav␤-like effect of CaMex on the channel gating.


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IP:

(a)

IgG FLAG

(b)

IgG FLAG

FLAGN-α1C 250 kDa IB:

IP:

anti-FLAG

(c)

IgG FLAG

50 kDa

ECFPN-CaM IB:

anti-α1C

anti-LC

Fig. S3. Specificity of the coimmunoprecipitation system for FLAG-␣1C and ECFPN-CaM. FLAGN-␣1C and ␣2␦ were coexpressed in COS1 cells with ECFPN-CaM. Proteins were co-immunoprecipitated (IP) with mouse IgG-agarose (Sigma) or FLAG m2 monoclonal affinity gel (Sigma), the immunoprecipitates were then eluted with 3xFLAG peptide and resolved by SDS/PAGE. Immunoblotting (IB) detection was carried out with anti-FLAG (a), anti-␣1C (b) or anti-LC Abs (c). Molecular mass calibration in kDa is shown at right. FLAG-␣1C and ECFPN-CaM were pulled down specifically only by anti-FLAG Ab.


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