Optimisation of Ca 2+ release assays

Many types of buffers and conditions have been used to perform Ca2+ release assays in various cell types. In order to optimise assay conditions and to show that free Ca2+ would not interfere with the reliability of the results a number of initial tests were carried out on the same sample of cells (MHN sample C1). Ca2+ release of fura 2 loaded B-lymphoblastoid cell line C1, triggered by 4-chloro-m-cresol (4CmC), was tested in the presence of buffer containing different concentrations of external Ca2+ and EGTA. The different conditions were as follows:

1. 1x BSS + 2 mM Ca2+

2. 1x BSS + 0.1 mM EGTA 3. 1x BSS + 1 mM EGTA 4. 1x BSS + 2 mM EGTA

The baseline fluorescence, representing the ratio of the emission at the two excitation wavelengths 340/380 nm, was recorded using a fluorometer for ~ 2 min before the addition of 900 µM 4-chloro-m-cresol (4CmC) that causes the 340/380 ratio of the two wavelengths to increase due to Ca2+ release from the stores, and subsequently the maximum amount of Ca2+ released was recorded. The difference between the baseline and the peak Ca2+ release (Δ) was recorded by subtracting the baseline ratio from the peak ratio (see figure 3.7) as well as the difference induced by the addition of 200 nM thapsigargin, an inhibitor of the Ca2+-ATPase SERCA, to provide an indication of the ER Ca2+ store size (table 3.2).

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Figure 3.7 Raw data of the 340/380 nm ratio change after the addition of 4CmC.

Average baseline values can be determined from the above graph for fura 2-AM loaded B- lymhoblastoid cells before 900 µM 4CmC was added to stimulate RYR1 Ca2+ _{release. While}

the ratio of 340/380 nm increases the peak Ca2+ release in B-lymphoblastoid cells can be determined. Subtracting the peak Ca2+ release from the baseline gives the Δ ratio for each individual cell line.

1 x BSS Δ ratio induced by 900 µM 4CmC Δ ratio induced by 200 nM thapsigargin + 2 mM Ca2+ 0.9 1.64 + 0.1 mM EGTA 0.75 1.7 + 1 mM EGTA 0.72 1.1 + 2 mM EGTA 0.2 0.5

Table 3.2 Influence of different Ca2+ concentrations in buffer on Ca2+ release in B- lymphoblastoid cells in response to 4CmC.

Addition of a high concentration of EGTA (2 mM) into Ca2+ _{imaging buffer leads to a}

decrease of mobile Ca2+ _{released by the addition of 4CmC. Furthermore the addition of high}

amounts of EGTA (1 and 2 mM) reduces the Ca2+ store size measured by the addition of thapsigargin.

This experiment was performed only once to assess the effects of external Ca2+ and EGTA on Ca2+ release from B-lymphoblastoid stores while the cells were only transferred into the measuring buffer just prior to measurements to prevent any effects the EGTA might have upon the cells. The results showed

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that the buffer containing 2 mM EGTA significantly reduces the releasable Ca2+ amount as well as the size of the Ca2+ containing stores. Even though RYR1 was activated with a high amount of 4CmC, Ca2+ released was negligible in buffer containing 2 mM EGTA (table 3.2). This effect was not as strong with 0.1 and 1 mM EGTA although it can be concluded that the Ca2+- containing stores appear to decrease in buffer containing 1 mM EGTA. Assays in BSS containing 0.1 and 1 mM EGTA gave almost the same Ca2+ release triggered by 4CmC as the 1 x BSS buffer containing 2 mM free Ca2+ (ratio ~0.8, see table 3.2). Therefore it was concluded that small amounts of free Ca2+ or EGTA do not significantly affect Ca2+ release assays. This experiment showed that it is important to use identical buffer conditions for every assay since changes in external Ca2+ concentration can influence the results of the assays. To assure that the EGTA does not damage the cells even before the assays were started, cells were kept in plus Ca2+ _{buffer until}

immediately before the experiment was carried out. The differences in ratio for 200 nM thapsigargin induced Ca2+ release is an indicator of the amount of Ca2+ stored in the ER since thapsigargin blocks SERCA leading to an indirect depletion of the ER stores. According to the data shown in table 3.2 there is almost no difference in the Ca2+ store size of B-lymphoblastoid cells measured in buffer containing 2 mM free Ca2+ (Δ ratio = 1.64) and 0.1 mM EGTA (Δ ratio = 1.7). Addition of 1 mM EGTA into 1 x BSS buffer diminished the Ca2+ store size to around 70 % (Δ ratio = 1.1 compared to assays in buffer containing 2 mM free Ca2+) while Ca2+ release assays performed in buffer containing 2 mM EGTA diminished the Ca2+ store size even further to around 30 % (Δ ratio = 0.5).

To further test the effect of external Ca2+ on 4CmC activation, concentration- response curves using 100–1000 µM 4CmC were obtained. The following combinations of Ca2+ and EGTA were used to measure concentration- response curves in the MHN cell line C1:

1. 1x BSS + 2 mM Ca2+

2. 1x BSS + 2 mM Ca2+ _{+ 2 mM EGTA (20 μM free Ca}2+₎

3. 1x BSS + 0.1 mM EGTA 4. 1x BSS + 2 mM EGTA

70 Results are shown in figure 3.8.

Figure 3.8 Ca2+ release assays for the MHN patient C1 in buffers containing different Ca2+ and EGTA concentrations in response to 4CmC.

Ca2+ release was calculated as a percentage of Ca2+ released by the addition of 1 mM 4CmC. Error bars represent the SEM (n= 5-8).

Ca2+ release was calculated as a percentage of Ca2+ released in response to the addition of 1 mM 4CmC and concentration-response curves were drawn using sigmoid curve fitting using the Origin 8.6 software. The deltas for all cell lines used in this study activated with 1 mM 4CmC are shown in appendix III. Figure 3.8 shows there is no significant difference (calculated using One-Way ANOVA with Bonferroni post hoc analysis) in the concentration-response curves for all buffers except for 1x BSS buffer containing 2 mM external Ca2+ which showed a decreased EC50 value (table 3.3).

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1x BSS buffer EC50 values (μM 4CmC) ±SEM

+ 2 mM Ca2+ _{560±12 *}

+ 2 mM Ca2+ + 2 mM EGTA

(20 μM free Ca2+) 652±14

+ 0.1 mM EGTA 665±11

+ 2 mM EGTA 681±17

Table 3.3 EC50 values for 4CmC stimulation obtained in different buffer conditions.

Significant differences were calculated using One-Way ANOVA and are indicated with an asterisk. EC50 are shown ±SEM (n=4-8).

The EC50 values calculated for buffer conditions 2, 3 and 4 are all within the

same range and do not show a significant difference compared to each other. A significant difference of EC50 values was obtained for conditions 2-4

compared to condition 1 (1x BSS containing 2 mM Ca2+ _{[p< 0.011]). Taking}

these results into account it was decided to use a buffer containing 20 µM free Ca2+ for further assays. For curve fitting a sigmoid model was chosen in order to represent concentration-response curves and calculate EC50 values

(table 3.3). Since EC50 represents the half maximal effective concentration of

an agonist, the lowest and the highest points of a sigmoid curve need to be defined to fit a sigmoid curve, in order to calculate EC50. The resulting fitted

curves do not pass through the 100 % value because the data set does not include enough data points to create a sigmoid model. The curve rather represents a polynomial distribution. In order to obtain better fitting sigmoid curves 4CmC concentrations >1 mM needed to be measured to obtain a value for the maximum Ca2+ release which would be shown by a plateau in the concentration-response curve. Using 4CmC concentrations above 1 mM can result in unspecific Ca2+ release due to SERCA inhibition and therefore store depletion resulting in non-reliable data [108]. Polynomial curve fitting can be used to obtain curves passing through the 100 % value but because EC50 values were to be calculated, sigmoid curves need to be fitted since

polynorminal curves do not represent any biological background. When using polynomial curves half maximal activation can be reported instead of EC50.

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3.3.5 Results for Ca2+ release assays in lymphoblastoid cell lines