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Spontaneous transient outward currents arise from microdomains where BK channels are exposed to a mean Ca(2+) concentration on the order of 10 microM during a Ca(2+) spark.

Zhuge R, Fogarty KE, Tuft RA, Walsh JV - J. Gen. Physiol. (2002)

Bottom Line: Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g((STOC))), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca(2+)]s.The Ca(2+) sparks did not change in amplitude over the range of potentials of interest.Moreover, given the constraints imposed by the estimated channel density and the Ca(2+) current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Imaging Group, Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

ABSTRACT
Ca(2+) sparks are small, localized cytosolic Ca(2+) transients due to Ca(2+) release from sarcoplasmic reticulum through ryanodine receptors. In smooth muscle, Ca(2+) sparks activate large conductance Ca(2+)-activated K(+) channels (BK channels) in the spark microdomain, thus generating spontaneous transient outward currents (STOCs). The purpose of the present study is to determine experimentally the level of Ca(2+) to which the BK channels are exposed during a spark. Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g((STOC))), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca(2+)]s. The Ca(2+) sparks did not change in amplitude over the range of potentials of interest. In contrast, the magnitude of g((STOC)) remained roughly constant from 20 to -40 mV and then declined steeply at more negative potentials. From this and the voltage dependence of BK channel activation, we conclude that the BK channels underlying STOCs are exposed to a mean [Ca(2+)] on the order of 10 microM during a Ca(2+) spark. The membrane area over which a concentration > or =10 microM is reached has an estimated radius of 150-300 nm, corresponding to an area which is a fraction of one square micron. Moreover, given the constraints imposed by the estimated channel density and the Ca(2+) current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.

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Ca2+ sparks recorded at −80 and 0 mV are of the same amplitude. (A) An example of a Ca2+ spark acquired at −80 mV in the presence of 1.8 mM extracellular Ca2+. Images show the spatial and temporal evolution of the Ca2+ spark. Top trace, the time course of change in fluorescence in the pixel (333 nm × 333 nm) where the peak fluorescence is reached, i.e., the epicenter pixel. Bottom trace, for the same spark, time course of Ca2+ signal mass, that is the total fluorescence for a volume subtended by an area 41 pixels on a side in the x-y plane and centered on the epicenter pixel. The signal mass is proportional to the total amount of Ca2+ released by the spark (see ZhuGe et al., 2000). (B, a) Amplitude histogram of Ca2+ sparks recorded at −80 and 0 mV and (b) their means. (c) Signal mass histogram of Ca2+ sparks recorded at −80 and 0 mV and (d) their means. (e) Mean frequency of Ca2+ sparks at −80 and 0 mV.
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fig2: Ca2+ sparks recorded at −80 and 0 mV are of the same amplitude. (A) An example of a Ca2+ spark acquired at −80 mV in the presence of 1.8 mM extracellular Ca2+. Images show the spatial and temporal evolution of the Ca2+ spark. Top trace, the time course of change in fluorescence in the pixel (333 nm × 333 nm) where the peak fluorescence is reached, i.e., the epicenter pixel. Bottom trace, for the same spark, time course of Ca2+ signal mass, that is the total fluorescence for a volume subtended by an area 41 pixels on a side in the x-y plane and centered on the epicenter pixel. The signal mass is proportional to the total amount of Ca2+ released by the spark (see ZhuGe et al., 2000). (B, a) Amplitude histogram of Ca2+ sparks recorded at −80 and 0 mV and (b) their means. (c) Signal mass histogram of Ca2+ sparks recorded at −80 and 0 mV and (d) their means. (e) Mean frequency of Ca2+ sparks at −80 and 0 mV.

Mentions: A possible reason for the decrease in g(STOC) at potentials more negative than −40 mV is that the amount of Ca2+ released from the SR during a spark decreases at negative potentials. We therefore examined Ca2+ sparks at 0 and −80 mV. The results are shown in Fig. 2. Fig. 2 A shows the temporal evolution of a typical Ca2+ spark, with images above and the traces indicating the fluorescence ratio and the signal mass below. Fig. 2 B, a, provides a histogram of the distribution of ΔF/Fo at 0 and −80 mV, and Fig. 2 B, c, does the same for the Ca2+ signal mass. Fig. 2 B, b, d, and e, compare the mean ΔF/F0, signal mass, and frequency at 0 and −80 mV. While spark frequency at 0 mV was greater, there was no difference in spark amplitude at 0 and −80 mV by either of the two measures of spark magnitude, ΔF/Fo or Ca2+ signal mass.


Spontaneous transient outward currents arise from microdomains where BK channels are exposed to a mean Ca(2+) concentration on the order of 10 microM during a Ca(2+) spark.

Zhuge R, Fogarty KE, Tuft RA, Walsh JV - J. Gen. Physiol. (2002)

Ca2+ sparks recorded at −80 and 0 mV are of the same amplitude. (A) An example of a Ca2+ spark acquired at −80 mV in the presence of 1.8 mM extracellular Ca2+. Images show the spatial and temporal evolution of the Ca2+ spark. Top trace, the time course of change in fluorescence in the pixel (333 nm × 333 nm) where the peak fluorescence is reached, i.e., the epicenter pixel. Bottom trace, for the same spark, time course of Ca2+ signal mass, that is the total fluorescence for a volume subtended by an area 41 pixels on a side in the x-y plane and centered on the epicenter pixel. The signal mass is proportional to the total amount of Ca2+ released by the spark (see ZhuGe et al., 2000). (B, a) Amplitude histogram of Ca2+ sparks recorded at −80 and 0 mV and (b) their means. (c) Signal mass histogram of Ca2+ sparks recorded at −80 and 0 mV and (d) their means. (e) Mean frequency of Ca2+ sparks at −80 and 0 mV.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2311396&req=5

fig2: Ca2+ sparks recorded at −80 and 0 mV are of the same amplitude. (A) An example of a Ca2+ spark acquired at −80 mV in the presence of 1.8 mM extracellular Ca2+. Images show the spatial and temporal evolution of the Ca2+ spark. Top trace, the time course of change in fluorescence in the pixel (333 nm × 333 nm) where the peak fluorescence is reached, i.e., the epicenter pixel. Bottom trace, for the same spark, time course of Ca2+ signal mass, that is the total fluorescence for a volume subtended by an area 41 pixels on a side in the x-y plane and centered on the epicenter pixel. The signal mass is proportional to the total amount of Ca2+ released by the spark (see ZhuGe et al., 2000). (B, a) Amplitude histogram of Ca2+ sparks recorded at −80 and 0 mV and (b) their means. (c) Signal mass histogram of Ca2+ sparks recorded at −80 and 0 mV and (d) their means. (e) Mean frequency of Ca2+ sparks at −80 and 0 mV.
Mentions: A possible reason for the decrease in g(STOC) at potentials more negative than −40 mV is that the amount of Ca2+ released from the SR during a spark decreases at negative potentials. We therefore examined Ca2+ sparks at 0 and −80 mV. The results are shown in Fig. 2. Fig. 2 A shows the temporal evolution of a typical Ca2+ spark, with images above and the traces indicating the fluorescence ratio and the signal mass below. Fig. 2 B, a, provides a histogram of the distribution of ΔF/Fo at 0 and −80 mV, and Fig. 2 B, c, does the same for the Ca2+ signal mass. Fig. 2 B, b, d, and e, compare the mean ΔF/F0, signal mass, and frequency at 0 and −80 mV. While spark frequency at 0 mV was greater, there was no difference in spark amplitude at 0 and −80 mV by either of the two measures of spark magnitude, ΔF/Fo or Ca2+ signal mass.

Bottom Line: Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g((STOC))), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca(2+)]s.The Ca(2+) sparks did not change in amplitude over the range of potentials of interest.Moreover, given the constraints imposed by the estimated channel density and the Ca(2+) current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Imaging Group, Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

ABSTRACT
Ca(2+) sparks are small, localized cytosolic Ca(2+) transients due to Ca(2+) release from sarcoplasmic reticulum through ryanodine receptors. In smooth muscle, Ca(2+) sparks activate large conductance Ca(2+)-activated K(+) channels (BK channels) in the spark microdomain, thus generating spontaneous transient outward currents (STOCs). The purpose of the present study is to determine experimentally the level of Ca(2+) to which the BK channels are exposed during a spark. Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g((STOC))), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca(2+)]s. The Ca(2+) sparks did not change in amplitude over the range of potentials of interest. In contrast, the magnitude of g((STOC)) remained roughly constant from 20 to -40 mV and then declined steeply at more negative potentials. From this and the voltage dependence of BK channel activation, we conclude that the BK channels underlying STOCs are exposed to a mean [Ca(2+)] on the order of 10 microM during a Ca(2+) spark. The membrane area over which a concentration > or =10 microM is reached has an estimated radius of 150-300 nm, corresponding to an area which is a fraction of one square micron. Moreover, given the constraints imposed by the estimated channel density and the Ca(2+) current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.

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Related in: MedlinePlus