Caffeine-induced release of intracellular Ca2+ from Chinese hamster ovary cells expressing skeletal muscle ryanodine receptor. Effects on full-length and carboxyl-terminal portion of Ca2+ release channels

Abstract

The ryanodine receptor (RyR)/Ca2+ release channel is an essential component of excitation-contraction coupling in striated muscle cells. To study the function and regulation of the Ca2+ release channel, we tested the effect of caffeine on the full-length and carboxyl-terminal portion of skeletal muscle RyR expressed in a Chinese hamster ovary (CHO) cell line. Caffeine induced openings of the full length RyR channels in a concentration-dependent manner, but it had no effect on the carboxyl-terminal RyR channels. CHO cells expressing the carboxyl-terminal RyR proteins displayed spontaneous changes of intracellular [Ca2+]. Unlike the native RyR channels in muscle cells, which display localized Ca2+ release events (i.e., "Ca2+ sparks" in cardiac muscle and "local release events" in skeletal muscle), CHO cells expressing the full length RyR proteins did not exhibit detectable spontaneous or caffeine-induced local Ca2+ release events. Our data suggest that the binding site for caffeine is likely to reside within the amino-terminal portion of RyR, and the localized Ca2+ release events observed in muscle cells may involve gating of a group of Ca2+ release channels and/or interaction of RyR with muscle-specific proteins.

Figure 5

Spatial and temporal patterns of caffeine-induced Ca²⁺ release in CHO cells expressing RyR. CHO cells expressing RyR-wt release Ca²⁺ upon exposure to caffeine. (A) Full frame confocal image of Fluo-3 fluorescence (Ca²⁺ activated) in CHO cells expressing the full length RyR: (a) control, (b) 0.5 s after addition of 10 mM caffeine, and (c) 5 min after washout of the caffeine. Increasing Ca²⁺-activated Fluo-3 fluorescence is indicated by the color bar on the right. Distance, in both x and y dimensions, is indicated by calibration bar indicating 10.0 μm. (B) Compressed line-scan image Fluo-3 fluorescence (proportional to [Ca²⁺]_i) of a single CHO cell exposed to 10 mM caffeine. Calcium rises rapidly and cooperatively (see small initial “foot” in rise), and declines slowly.

Figure 6

Dose-dependent effect of caffeine on CHO cells expressing RyR-wt. Fluorescence ratio, derived from one selected pixel of line scan images such as those in Fig. 5 B, is plotted as a function of time. The pixel size was 0.2 μm. Results are from three different CHO cells. Doses of caffeine: (A) 0.5, (B) 2.0, and (C) 10 mM. The rate of rise in fluorescence ratio ([Ca²⁺]) is highly dose dependent, possibly indicating dose-dependent cooperativity of the opening of the Ca²⁺ release channels. The fact that the peak fluorescence ratio reached was greatest in 2.0 mM caffeine may be the result of variability in the amount of Ca²⁺ in the intracellular stores of different cells. The number of cells tested are n = 2 (0.1 mM), n = 5 (0.2 mM), n = 4 (0.5 mM), n = 4 (2 mM), n = 2 (5 mM), and n = 10 (10 mM).

Figure 7

Lack of Ca²⁺ sparks in CHO cells expressing RyR-wt. (A) Line-scan image of fluorescence ratio (Fluo-3) in c1148 cells under control conditions. No local calcium transients or sparks are evident. (B) Line-scan image of c1148 cells during the initial rise in [Ca²⁺]_i in the presence of 0.5 mM caffeine. [Ca²⁺] rises very slowly from resting levels, but no sparks are evident. (C) Typical spontaneous Ca²⁺ sparks in a cardiac myocyte. All line-scan images were obtained and presented at exactly the same spatial and temporal resolution, with the same confocal microscope.

Figure 8

Spontaneous oscillation of intracellular Ca²⁺ in CHO cells expressing RyR-C. (A) Sequence of 16 full-frame images of Ca²⁺- activated Fluo-3 fluorescence taken at 20-s intervals. (B) Plot of Ca²⁺-activated Fluo-3 fluorescence as a function of time in the cell illustrated in A. The existence of spontaneous oscillations of [Ca²⁺]_i may explain the apparent high resting [Ca²⁺]_i in population measurements of [Ca²⁺]_i in CHO cells expressing RyR-C.

Figure 1

Heterologous expression of skeletal muscle RyR in CHO cells. Western blot analysis of RyR proteins expressed in Chinese hamster ovary cells. Total cell lysates were run on a 3–12% gradient SDS polyacrylamide gel, and blotted with monoclonal antibody (RR2) against the rabbit skeletal muscle RyR (Bhat et al., 1997). Lane 1, SR vesicles from rabbit skeletal muscle; lane 2, untransfected CHO cells; lanes 3 and 6, COOH-terminal RyR proteins (∼130 kD) stably expressed in CHO cells (ryr-c); lane 4, CHO cells transfected with GFP-RyR-C; lane 5, CHO cells transfected with GFP-RyR; lane 7, full length RyR (∼560 kD) stably expressed in CHO cells (c1148).

Figure 2

Localization of RyR in intracellular membranes of CHO cells. Confocal measurement of green fluorescence in CHO cells transfected with (A) pEGFP-N1, (B) pcDNA3 (GFP-RyR), and (C) pcDNA3 (GFP-RyR-C). Cells expressing GFP alone exhibit a diffuse pattern of fluorescence throughout the cell (A), whereas cells expressing the GFP-RyR and GFP-RyR-C fusion proteins show localized fluorescence signal near the perinuclear region of the cells (B and C). The panels from left to right show representative sections through the cells at 0.5-μm intervals. The cells have an average thickness of ∼10 μm.

Figure 3

Effect of caffeine on single RyR-wt and RyR-C channels. Selected single channel currents at +50 mV from a continuous experiment with the RyR-wt (A) and RyR-C (B) channels incorporated into the lipid bilayer membrane. Under control conditions (220 μM [Ca²⁺]_i), the RyR-wt had an average Po of 0.121 ± 0.044 (n = 19), and the RyR-C channel had an average Po of 0.201 ± 0.036 (n = 12). The addition of 10 mM EGTA lowered the free [Ca²⁺] to 0.08 μM and reduced the Po of RyR-wt to 0.013 ± 0.002 (n = 5), and Po of RyR-C to 0.008 ± 0.003 (n = 4). Subsequent addition of 10 mM caffeine increased the Po of RyR-wt to 0.198 ± 0.063 (n = 4). Po of the RyR-C channel remained unchanged after the addition of 10 mM caffeine (Po = 0.008 ± 0.002, n = 4).

Figure 4

Caffeine-induced release of Ca²⁺ in CHO cells. Changes in [Ca²⁺]_i were measured in cells (10⁶/ml) loaded with Fura-2 AM and maintained under continuous stirring in a cuvette. (A) Application of 10 mM caffeine (Caf) does not induce release of intracellular Ca²⁺ in untransfected CHO cells, whereas thapsigargin (Thg, 500 nM) causes a rise in [Ca²⁺], suggesting intact intracellular Ca²⁺ store in these cells. (B) In c1148 cells, an increase in [Ca²⁺]_i from 0.192 ± 0.038 μM (n = 4) to 1.222 ± 0.257 μM (n = 3) is seen after stimulation with caffeine. (C) CHO cells expressing RyR-C do not respond to stimulation with caffeine. The resting intracellular [Ca²⁺] in these cells is 0.422 ± 0.071 μM (n = 7). All fluorescence traces are representative of at least three separate experiments. Gaps in the recordings are due to the opening of the compartment to make additions.