Calcium Uptake in Crude Tissue Preparation

SUMMARY

The various isoforms of the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) are responsible for the Ca²⁺ uptake from the cytosol into the endoplasmic or sarcoplasmic reticulum (ER/SR). In some tissues, the activity of SERCA can be modulated by binding partners, such as phospholamban and sarcolipin. The activity of SERCA can be characterized by its apparent affinity for Ca²⁺ as well as maximal enzymatic velocity. Both parameters can be effectively determined by the protocol described here. Specifically, we describe the measurement of the rate of oxalate-facilitated ⁴⁵Ca uptake into the SR of crude mouse ventricular homogenates. This protocol can easily be adapted for different tissues and animal models as well as cultured cells.

1. Introduction

The ability to mobilize Ca²⁺ has been, for decades, a well-established characteristic of ER/SR microsomes [1–3]. This observation was of particular importance in muscle tissue where Ca²⁺ is the trigger for contraction [4, 5]. This Ca²⁺ uptake activity was later determined to be produced by an integral membrane ATPase protein, SERCA[6–8]. Further research identified phospholamban [9, 10], and later sarcolipin [11, 12], as inhibitors of SERCA activity in cardiac and skeletal muscles, respectively.

SERCA activity follows traditional Michaelis-Menten enzyme kinetics [13] described by the equation

$$\nu =\frac{V_{\mathit{\text{max}}}[S]}{K_m+[S]}$$

Here v is the observed rate and [S] is the concentration of rate-limiting substrate, which is Ca²⁺ in this case. In other words, the rate of Ca²⁺ binding to SERCA is the determinant of SERCA activity. (ATP is also a critical substrate for SERCA, but generally is well above saturating concentrations and therefore not rate limiting and not included in basic descriptions of SERCA activity). V_max is the maximal rate of the enzyme and K_m is the Michealis constant which corresponds to the substrate concentration at the half maximal rate. K_m is an inverse measure of substrate affinity, meaning that a low value corresponds to a high affinity and vice versa. In simpler terms – at low Ca²⁺ concentrations, where little is available to be pumped across the membrane, SERCA activity is low. Elevating Ca²⁺ increases SERCA activity up to a maximal level.

The Ca²⁺ uptake protocol described here produces the Michaelis-Menten response curve of SERCA over the physiologically relevant Ca²⁺ concentrations (10 nM to 10 μM), which also corresponds to the full dynamic range of the enzyme [10] (Figure 1). For easy visualization and discussion, Ca²⁺ concentrations are represented with pCa values, which correspond to the inverse log of the molar concentration.

Figure 1. Representative SERCA activity curves following Michaelis-Menten enzyme kinetics.

(A) V_max describes the maximal uptake rate of the sample and K_m indicates the Ca²⁺ concentration at half the maximal rate. (B) The dashed lines showing downward and leftward shifts from the solid black line correspond to a decrease maximal rate (V_max) and an increase in Ca²⁺ affinity (K_m), respectively. For cardiac preparations, a downward shift would correspond to the diminished SERCA protein expression as observed in heart failure. The leftward shift would indicate relief of PLN inhibition either through phosphorylation or ablation.

Changes in PLN or SLN inhibition will generate a leftward or rightward shift in this activity curve signifying an altered Ca²⁺ affinity. V_max typically varies depending on SERCA expression level. Variations in K_m and V_max will also vary between species, tissue type and SERCA isoform.

The protocol presented here is a detailed description of our standard laboratory procedure [14–20] and is adapted from the Millipore filtration technique [21]. In principle, this assay measures the amount of ⁴⁵Ca retained in homogenate microsomes over time after being transported by SERCA. These microsomes are collected by a nitrocellulosse membrane and subsequently washed to allow excess Ca²⁺ that is not sequestered by the microsomes to pass through. Ruthenium Red blocks extrusion of Ca²⁺ out of the microsomes through ion channels [22] and prevents uptake into the mitochondria [23]. Ca²⁺ precipitates with oxalate inside ER/SR microsomes [24–26], which serves multiple purposes in this assay. First, this precipitation lowers the free Ca²⁺ inside the microsomes, which eliminates the generation of a concentration gradient that would slow SERCA activity over time thereby allowing consistent Ca²⁺ transport for the duration of the assay [27, 28]. Secondly, it further prevents Ca²⁺ extrusion out of the microsomes. Oxalate also preferentially accumulates in ER/SR microsomes via a non-specific anion transporter [24–26, 29]. Therefore, the oxalate trapped Ca²⁺ resides in only ER/SR microsomes which eliminates the need for ER/SR purification that may introduce significant variability between samples.

It is important to note that this assay describes the initial rates of steady-state activity of SERCA [27], although the cytosolic environment is not at steady-state. Increased SERCA activity decreases cytosolic Ca²⁺ thereby decreasing its own enzymatic activity.

2. Materials

2.1. Solutions

Prepare all stock solutions using ultrapure water and analytical grade reagents and store at 4°C unless otherwise noted.

1. Homogenization Buffer: Prepare on the day of the experiment according to Table 1 and keep on ice until use.

2. Reaction Mixture: Prepare on the day of the experiment according to Table 2 and keep on ice until use.

3. 0.1 M ATP: For 75 ml, dissolve 4.27 g ATP (MW 569.1 g/mol) in 40 ml of H₂O and adjust the pH to 7.0 using 1 N NaOH. Keep on ice. Bring the volume to 70 ml. Calculate the true concentration by measuring and averaging the absorbance at 259 nm of multiple dilutions (1:1000 to 1:4000). M = Abs at 259 nm/15.4 × 10³. Dilute the sample to exactly 0.1 M, aliquot, and store at −80°C.

4. 1.14 × 10⁻⁴ M Ruthenium Red: The day of the experiment, dilute approximately 0.1 mg in 1 ml of water. Calculate the true concentration by measuring the absorbance at 533 nm at multiple dilutions (1:100 to 1:300). M = Abs at 533 nm/6.4×10⁴. Dilute the sample to 1.14 × 10⁻⁴ M. 400 μl are needed for each assay in duplicate.

5. ⁴⁵Ca: Prepare an initial stock of ⁴⁵Ca to a concentration of 2.5 μCi/μl in H₂O. For each assay in duplicate, 900 μl of 40 μCi/ml (36 μCi) is needed. 36 μCi corresponds to 14.4 μl of a 2.5 μCi/μl stock. To account for decay, divide 14.4 μl by the decay factor obtained from a ⁴⁵Ca decay chart. Add H₂O to bring final volume to 900 μl.

6. 10.00 mM CaCl₂: Either purchase an analytical grade calcium solution or have the concentration of a prepared stock analytically verified.

7. Wash Buffer: 20 mM Tris-HCl, 2 mM EGTA, pH 7.0.

8. Tissue of interest: This assay is optimized for whole mouse ventricular cardiac tissue (approximately 20 mg) and can be adapted for other tissue types or cultured cell lines. The abundance of SERCA protein, which is high in muscle, should be taken into account when adapting to non-muscle tissue or cells.

Table 1.

Homogenization Buffer

Stock	[Final]	Amount for 5 ml
0.5 M phosphate buffer, pH 7.0	50 mM	0.5 ml
0.1 M NaF	10 mM	0.5 ml
0.5 M EDTA	1 mM	0.01 ml
Sucrose	0.3 M	0.52 g
0.02 M PMSF (−20°C)	0.3 mM	0.075 ml
0.1 M DTT (−20°C)	0.5 mM	0.0125 ml

Table 2.

Reaction Mixture

Stock	[Final]	Amount for 30 Reactions
0.08 M Imidazol, pH 7.0	40 mM	22.5 ml
1 M KCl	95 mM	4.275 ml
0.1 M NaN3	5 mM	2.25 ml
0.1 M MgCl2	5 mM	2.25 ml
0.5 M EGTA	0.5 mM	0.045 ml
0.1 M K+ oxalate	5 mM	2.25 ml

2.2. Equipment

1. Vacuum filtration system

2. 0.45 μm nitrocellulose Millipore filters

3. Water bath set to 37°C

4. Reaction tubes: 15×85 mm borosilicate glass culture tubes

5. 20 ml scintillation vials

6. Scintillation counter

7. Tissue homogenizer

8. Vortex

3. Methods

3.1. Uptake Reaction

The key to this assay is consistent pipetting and great care should be taken to yield accurate and precise results. To further enhance accuracy, we recommend performing the entire assay in duplicate. Also, start at the lowest Ca²⁺ concentration and move to higher ones. On the duplicate set of reactions, start at the highest Ca²⁺ concentration and move to lower ones.

1. Set up the 13 reaction tubes in duplicate (26 total).

2. Set up 5 scintillation vials for each reaction: 1 blank, 1 standard, and 1 for each of the 3 time points (130 total).

3. Prepare reaction tubes according to Table 3.

4. Homogenize tissue in homogenization buffer and keep on ice until used for each reaction. 75 μl is necessary for each of the 26 reactions (1.950 ml total). Approximate protein concentration should be between 0.5 and 5.0 mg/ml. Final rates will be normalized to quantified concentration.

5. Place four 0.45 um Millipore filters on filtration manifold for the first reaction tube.

6. Wash each filter 2 times with 2.5 ml Wash Buffer.

7. Add 13.2 μl of 0.114 mM Ruthenium Red (1 μM final) to first reaction tube.

8. Add 75 μl of homogenates to the first reaction tube and slightly vortex (avoiding air bubble formation) to thoroughly mix.

9. Place the reaction tube in a 37°C water bath.

10. After 30 seconds remove an aliquot of 290 ul for nonspecific binding (also called blank). Pass the aliquot through a 0.45 μm Millipore membrane.

11. Initiate the reaction by adding 60 μl 0.1 M ATP, [Final] = 5 mM. Mix thoroughly with the pipet.

12. Take out 300 μl at 30, 60, and 90 second time points and pass each aliquot through a different membrane.

13. Wash each membrane 2 times with 2.5 ml Wash Buffer.

14. Place the filters in the scintillation vials.

15. Repeat steps 5–14 for the remaining reaction tubes.

16. When finished, add 60 μl from the remaining reaction mixture to the standard scintillation vial for each reaction.

17. Add 10 ml of scintillation fluid to each scintillation vial.

18. Measure samples with a scintillation counter.

Table 3.

Calcium Dilutions

			Volume to Add (μl)
Buffer #	pCa	10.00 mM CaCl2	dH2O	45Ca (40 μCi/ml)	Reaction Mixture
1	8	2.6	228.3	1.9	1119
2	7.5	7.7	219.4	5.7	1119
3	7.2	13.9	208.7	10.2	1119
4	7	19.9	198.3	14.6	1119
5	6.8	27.4	185.2	20.2	1119
6	6.6	36.0	170.3	26.5	1119
7	6.4	44.8	155.0	33.0	1119
8	6.3	49.1	147.6	36.1	1119
9	6.2	53.2	140.5	39.1	1119
10	6	60.5	127.8	44.5	1119
11	5.8	66.8	116.9	49.1	1119
12	5.5	74.7	103.1	55.0	1119
13	5	89.9	76.8	66.1	1119

3.2. Data Analysis

1. Quantify homogenate protein concentration using the Bradford method.

2. The scintillation counter yields values in counts per minute (cpm) which can be converted to moles of Ca²⁺ using the standard sample from step 16 in section 3.1. This sample has not been filtered and therefore is representative of the total Ca²⁺ in each reaction mixture. The amount of microsome sequestered Ca²⁺ is represented by the following equation

   $$\mathit{\text{Ca Uptake}}(\mathit{\text{nmol}})=\frac{\mathit{\text{sample cpm}}-\mathit{\text{blank cpm}}}{\mathit{\text{standard cpm}}\times 5}\times \mathit{Ca}(\mathit{\text{nmol}})$$

   Here Ca (nmol) represents the amount of cold Ca²⁺ present in each aliquot (300 μl) of each individual reaction mixture (Table 3: 10.00 mM CaCl₂ column) which is one-fifth of the amount in the total reaction mixture (1.5 ml).

3. After all Ca-uptake values are calculated, perform a linear regression from the 3 times points (30, 60, and 90 seconds) for each pCa. The slope of this line corresponds to the rate of Ca²⁺ uptake in nmol/min. These values should then be normalized to the amount of protein giving a final normalized rate in nmol/min/mg (Figure 2A).

4. Plot the average of the duplicate calculations against the corresponding Ca²⁺ concentration. This plot can then be fit with the Hill equation to determine the V_max and K_m parameters (Figure 2B).

Figure 2. Examples plots of the initial rates of SERCA activity from C57/BL6 mouse ventricular homogenates.

(A) The rate at each Ca²⁺ concentration is determined by calculating the slope from the Ca-uptake values from the 3 time points. These rates can then be plotted against the Ca²⁺ concentration (B) and fit with the Hill equation to determine the V_max and K_m parameters. For this example plot, V_max = 80.2 ± 0.5 and K_m = pCa 6.468 ± 0.007 (340 nM).