Depression of voltage-activated Ca²⁺ release in skeletal muscle by activation of a voltage-sensing phosphatase

Voltage-activated Ca²⁺ release in VSP-expressing fibers. (A) Line-scan images of depolarization-induced Ca²⁺ release in a VSP-negative fiber (left) and in a Ci-VSP–expressing fiber (right); fibers were depolarized from −80 to 10 mV (traces on top). (B) Ca²⁺ transients (rhod-2 F/F₀, thick traces) and corresponding rate of Ca²⁺ release (thin red traces) in response to pulses from −80 mV to the indicated values. (C) Peak Ca²⁺ release versus voltage in VSP-negative fibers (n = 9) and in Ci-VSP–expressing fibers (n = 6). Error bars represent ± SEM. (D) Average Ca²⁺ release in response to a pulse from −80 to 10 mV in VSP-negative fibers (line) and in Ci-VSP–expressing fibers (blue squares).

Ca²⁺ transients in VSP-expressing fibers depolarized by large pulses. (A) Voltage-pulse protocol. (B and C) Corresponding F/F₀ rhod-2 traces in a Ci-VSP–negative and in a Ci-VSP–positive fiber, respectively. Arrows point to the F/F₀ level 3 s after the onset of the pulse. (D) Mean values for the ratio of the initial peak rhod-2 Ca²⁺ transient in response to the pulse to 120 mV to the initial peak value in response to the pulse to 20 mV in VSP-negative (n = 11) and Ci-VSP–positive fibers (n = 8). (E) Mean values for the ratio of the rhod-2 Ca²⁺ level 3 s after the onset of the pulse to 120 mV to the corresponding value during the pulse to 20 mV. Error bars represent ± SEM. *, P ≤ 0.05.

Ca²⁺ transients in response to trains of depolarizing steps in VSP-negative fibers and Ci-VSP–expressing fibers. (A) Control and test voltage-pulse protocols. (B and D) Rhod-2 F/F₀ traces recorded in response to the protocols shown in A in a VSP-negative fiber and in a Ci-VSP–expressing fiber, respectively. The graphs on the right show the ratio of the successive peak F/F₀ values during the test record to the corresponding values during the control record, plotted versus the pulse number during the protocols; values were normalized to the ratio value corresponding to the first Ca²⁺ transient. (C and E) Ca²⁺ release traces calculated from the above-corresponding rhod-2 transients. The graphs on the right show the ratio of the successive peak Ca²⁺ release values during the test record to the corresponding values during the control record, plotted versus the pulse number; values were normalized to the ratio value corresponding to the first Ca²⁺ transient. (F; left) Mean values for the normalized ratio of peak F/F₀ during the test record to the corresponding peak F/F₀ during the control record, versus the pulse number, in VSP-negative and in Ci-VSP–positive fibers. (Right) Corresponding mean values for the normalized ratio of peak Ca²⁺ release during the test record to the corresponding peak Ca²⁺ release during the control record. Error bars represent ± SEM.

Membrane current during trains of depolarizing steps in VSP-negative fibers and Ci-VSP–expressing fibers. Mean value for the membrane current measured at the end of each depolarizing pulse during the control and test protocols shown in Fig. 4. The horizontal axis is positioned at the zero-current level. Error bars represent ± SEM.

Reversibility of the effect of VSP activation on trains of Ca²⁺ transients. (A) Pulse protocols that were successively applied. (B) F/F₀ rhod-2 traces elicited in a Ci-VSP–expressing fiber challenged by the protocols shown in A. (C) Mean values for the peak F/F₀ rhod-2 signals during the test 1, control 2, and test 2 records (n = 12 Ci-VSP–expressing fibers); in each fiber, successive peak F/F₀ values were divided by the corresponding values for the same pulse number during the control 1 record. Error bars represent ± SEM.

Ca²⁺ transients in response to trains of depolarizing steps in Dr-VSP–expressing fibers. (A) Control and test voltage-pulse protocols. (B) Rhod-2 F/F₀ traces recorded in response to the protocols shown in A in a Dr-VSP–positive fiber. (C) Mean values for the normalized ratio of peak F/F₀ during the test record to the corresponding peak F/F₀ during the control record, versus the pulse number in Dr-VSP–positive fibers (n = 7). Mean values for VSP-negative fibers are the same as in Fig. 4. Error bars represent ± SEM.

Ca²⁺ current after strong depolarizing pulses in Ci-VSP–expressing fibers. In each panel, top traces show the two voltage-pulse protocols that were applied to the fiber; the protocol with prepulse to 120 mV (test) was bracketed by two protocols with prepulse to 20 mV (controls). (A–C) Superimposed membrane current records in response to the above shown protocols. Currents recorded during the two control protocols are in black, whereas current elicited by the test protocol is presented in red. In A, records are presented at both low (top) and high magnification (bottom). In B and C, only the high magnification views are presented. (D) Mean values for the ratio of Ca²⁺ current amplitude after the prepulse to 120 mV to its amplitude after the prepulse to 20 mV; values are plotted versus the pulse interval between the pre- and test pulses. Results are from seven VSP-negative and eight Ci-VSP–positive fibers. Error bars represent ± SEM.

PLCδ₁PH-mRFP fluorescence in fibers expressing Ci-VSP. (A) Confocal images of a portion of a muscle fiber expressing Ci-VSP using the pIRES-EGFP plasmid and PLCδ₁PH-mRFP. The image on the right shows the PLCδ₁PH-mRFP fluorescence at higher magnification, and the graph below shows the fluorescence profile along the longitudinal axis of the highlighted rectangular region (yellow box). (B) Time course of change in PLCδ₁PH-mRFP fluorescence averaged over a given area of the fiber while applying 5-s-long depolarizing pulses according to the protocol shown on top (closed circles); superimposed time course of change in PLCδ₁PH-mRFP fluorescence measured subsequently over a nearby fiber area while no pulse was applied (open circles); synthetic trace generated from a two-exponential fit to the closed-circles record, using only the 20 first and 20 last values in the record for the fit (line). (C) Changes in fluorescence associated with the depolarizing pulse protocol after normalization by the record taken when no pulse was applied (closed circles); changes in fluorescence associated with the depolarizing-pulse protocol after normalization by the synthetic record (open circles).

Spatially segregated distinct changes in PLCδ₁PH-mRFP fluorescence upon strong membrane depolarization in fibers expressing Ci-VSP. (A) Confocal images of a portion of a muscle fiber expressing Ci-VSP and PLCδ₁PH-mRFP. Images from left to right were taken before, during, and after a 5-s-long depolarizing pulse to 50 mV, respectively. The graph below each image shows the fluorescence intensity profile along the rectangular area highlighted in the image on the left. (B; left graph) Difference between the intensity at a given location of the fluorescence profile during the pulse (Fluo+50) and its corresponding intensity before the pulse (Fluo−80(1)) versus Fluo−80(1). (Right graph) Difference between the intensity at a given location of the fluorescence profile during the pulse (Fluo+50) and its corresponding intensity after the pulse (Fluo−80(2)) versus Fluo−80(2). (C) Result of the analysis similar to that shown in Fig. 9 C, except that pixels with high initial intensity and low initial intensity were separated; pixel selection was achieved by adjusting manually the threshold tool in ImageJ in the first image of the sequence. (D) Mean relative peak change in PLCδ₁PH-mRFP fluorescence versus membrane potential calculated from three fibers that experienced the pulse protocol shown in C; closed circles, open squares, and open circles correspond to values obtained using all, high intensity, and low intensity initial pixels, respectively. Error bars represent ± SEM.