Microdomain [Ca²⁺] near ryanodine receptors as reported by L-type Ca²⁺ and Na+/Ca²⁺ exchange currents

Abstract

During Ca²⁺ release from the sarcoplasmic reticulum triggered by Ca²⁺ influx through L-type Ca²⁺ channels (LTCCs), [Ca²⁺] near release sites ([Ca²⁺]nrs) temporarily exceeds global cytosolic [Ca²⁺]. [Ca²⁺]nrs can at present not be measured directly but the Na+/Ca2+ exchanger (NCX) near release sites and LTCCs also experience [Ca²⁺]nrs. We have tested the hypothesis that ICaL and INCX could be calibrated to report [Ca²⁺]nrs and would report different time course and values for local [Ca²⁺]. Experiments were performed in pig ventricular myocytes (whole-cell voltage-clamp, Fluo-3 to monitor global cytosolic [Ca²⁺], 37◦C). [Ca²⁺]nrs-dependent inactivation of ICaL during a step to +10 mV peaked around 10 ms. For INCX we computationally isolateda current fraction activated by [Ca²⁺]nrs; values were maximal at 10 ms into depolarization. The recovery of [Ca²⁺]nrs was comparable with both reporters (>90% within 50 ms). Calibration yielded maximal values for [Ca²⁺]nrs between 10 and 15 μmol l⁻¹ with both methods. When applied to a step to less positive potentials (-30 to -20 mV), the time course of [Ca²⁺]nrs was slower but peak values were not very different. In conclusion, both ICaL inactivation and INCX activation, using a subcomponent analysis, can be used to report dynamic changes of [Ca²⁺]nrs. Absolute values obtained by these different methods are within the same range, suggesting that they are reporting on a similar functional compartment near ryanodine receptors. Comparable [Ca²⁺]nrs at +10 mV and -20 mV suggests that, although the number of activated release sites differs at these potentials, local gradients at release sites can reach similar values.

Figure 1. Extent and time course of inactivation of I_CaL related to SR Ca²⁺ release

A, protocol and typical example of I_CaL and Ca²⁺ transients with increasing SR Ca²⁺ load. After depletion of SR Ca²⁺ with caffeine, cells were reloaded by applying repetitive depolarizing pulses to +10 mV in 0 Na⁺ conditions to prevent Ca²⁺ removal on NCX. The arrow on I_CaL indicates the increasing rate of inactivation of I_CaL. Current traces (right) corresponding to a low (filled circle) or large (open circle) amount of SR Ca²⁺ release are shown after normalization to I₀, the first I_CaL. B, difference current between normalized traces from A, together with the Ca²⁺ transient of the fourth pulse. C, difference current superimposed on the SR release flux calculated from the Ca²⁺ transient of the fourth pulse (see text for details).

Figure 2. I_CaL and release-dependent modulation at different levels of depolarization

A, examples of Ca²⁺ transients in confocal line scan recordings with corresponding I_CaL during a step to +10 and −30 mV. B, traces of I_CaL, Ca²⁺ transients and release-dependent inactivation at −20 mV (as in Fig. 1). C, time course of release-dependent inactivation of I_CaL at different voltages; averaged traces of difference currents from n = 11 cells at +10 mV and n = 6 cells at −20 mV. D, comparison of maximal inactivation and time to peak of the difference currents recorded at +10 (n = 11) and −20 mV (n = 6).

Figure 3. SR Ca²⁺ release-dependent inactivation and recovery of I_CaL in the presence of Na⁺/Ca²⁺ exchange

A, voltage clamp protocol, current recordings and corresponding Ca²⁺ transients. A step to +10 mV from the holding potential is given to set a reference for maximal availability of LTCCs (peak I_CaL is I₀, marked by arrow, no inactivation). Subsequently a series of depolarizing steps to −30 mV are applied. Each step is interrupted with a test step to +10 mV at increasing time intervals after the initial depolarization, starting at 10 ms, with increments of 10 ms. With these test steps we measure availability of Ca²⁺ channels (peak of I_CaL, I) and thus the inactivation of LTCCs incurred during the step to −30 mV, compared to the reference, I₀; the degree of inactivation is expressed as 1 –I/I₀. A test step at 50 ms is marked in blue (1 –I/I₀ is 0.21), a step at 100 ms is marked in red (1 –I/I₀ is 0.11). B, left panel, mean data obtained with the protocol of panel A for degree of inactivation of LTCCs during a step to −30 mV (n = 8). The right panel repeats the data from Fig. 2C for a step to −20 mV for comparison.

Figure 4. Estimation of [Ca²⁺]_nrs from release-dependent inactivation of I_CaL

A, [Ca²⁺]_nrs estimated using the calibration curve for steps to +10 and −20 mV (data from Fig. 2C and using calibration of Hofer et al. (1997)). B, protocol for establishing an intracellular calibration curve. After the first pulse evoking a reference I_CaL (I₀, no inactivation), 10 mm caffeine was applied to the cell to induce SR Ca²⁺ release. During the declining phase of the [Ca²⁺]_i transient a test depolarizing pulse was given to record I_CaL (I) and estimate Ca²⁺-dependent inactivation. This was applied with different delays to obtain I at different [Ca²⁺]. C, Ca²⁺-dependent inactivation of I_CaL plotted against [Ca²⁺]. Maximal inactivation (M) and Ca²⁺ causing half-maximal inactivation (K_d) were estimated for individual cells by fitting the equation 1 –I/I₀=M× Ca/(Ca +K_d) to the data points. A calibration curve to relate release-dependent inactivation and Ca²⁺ was constructed using the mean values of M and K_d (0.61 ± 0.025 and 780 ± 67 nm, respectively, n = 10 cells). D, [Ca²⁺]_nrs estimated using the calibration curve for steps to +10 and −20 mV (data from Fig. 2C).

Figure 5. Translating NCX current into [Ca²⁺]_nrs using the global NCX current

A, voltage protocol illustrating how a step to +10 mV is interrupted by repolarization to −70 mV at increasing time intervals, to record the tail NCX currents on repolarization. The letters a, d, and h denote the corresponding repolarization time and currents. The current traces during the depolarization have been omitted for clarity. B, calibration for converting NCX current amplitude to [Ca²⁺]. Typical recordings of Ca²⁺ transient and the concomitant inward I_NCX after application of 10 mm caffeine. A linear relationship (right) between I_NCX amplitude and Ca²⁺ was obtained by fitting a line to the declining phase of the caffeine induced Ca²⁺ transient (intercept 106.5 ± 14.1 nm, slope –0.00194 ± 0.000129 pA pF⁻¹ nm⁻¹, n = 25). C, global Ca²⁺ transient (filled circles) during SR Ca²⁺ release and subsarcolemmal Ca²⁺ (open circles) estimated by using the linear relationship in B for a step to +10 mV (left panel, n = 4), and to −30 mV (right panel, n = 8).

Figure 6. NCX tail current analysis with two compartments to extract [Ca²⁺]_nrs

A, two recordings at 10 ms and at 30 ms into the depolarizing step are shown with the global [Ca²⁺]_i transient and the recorded NCX tail current on repolarization (black current trace). Predicted values for NCX activated by the cytosolic Ca²⁺ transient are calculated using the NCX model equation of (Weber et al. 2001) (green trace). The difference current between the recorded (black trace) and calculated (green trace) then reflects NCX activated by Ca²⁺ near release sites and not reported by the cytosolic dye. This measurement is repeated every 10 ms. B, [Ca²⁺]_nrs for during the example depolarizing step to +10 mV as calculated from the values of local NCX current at the corresponding time points.

Figure 7. Time course of [Ca²⁺]_nrs as estimated from the NCX tail currents

A, measured total (filled circles) NCX currents and calculated extra NCX (open circles) at +10 (n = 4) and −30 mV (n = 8). B, calculated [Ca²⁺]_nrs (open circles) and measured global [Ca²⁺] (filled circles) during steps to +10 (n = 4) and −30 mV (n = 8).