Ca(2+) spark sites in smooth muscle cells are numerous and differ in number of ryanodine receptors, large-conductance K(+) channels, and coupling ratio between them.
Ca(2+) sparks are highly localized Ca(2+) transients caused by Ca(2+) release from sarcoplasmic reticulum through ryanodine receptors (RyR). In smooth muscle, Ca(2+) sparks activate nearby large-conductance, Ca(2+)-sensitive K(+) (BK) channels to generate spontaneous transient outward currents (STOC). The properties of individual sites that give rise to Ca(2+) sparks have not been examined systematically. We have characterized individual sites in amphibian gastric smooth muscle cells with simultaneous high-speed imaging of Ca(2+) sparks using wide-field digital microscopy and patch-clamp recording of STOC in whole cell mode. We used a signal mass approach to measure the total Ca(2+) released at a site and to estimate the Ca(2+) current flowing through RyR [I(Ca(spark))]. The variance between spark sites was significantly greater than the intrasite variance for the following parameters: Ca(2+) signal mass, I(Ca(spark)), STOC amplitude, and 5-ms isochronic STOC amplitude. Sites that failed to generate STOC did so consistently, while those at the remaining sites generated STOC without failure, allowing the sites to be divided into STOC-generating and STOC-less sites. We also determined the average number of spark sites, which was 42/cell at a minimum and more likely on the order of at least 400/cell. We conclude that 1) spark sites differ in the number of RyR, BK channels, and coupling ratio of RyR-BK channels, and 2) there are numerous Ca(2+) spark-generating sites in smooth muscle cells. The implications of these findings for the organization of the spark microdomain are explored.
Department of Physiology, University of Massachusetts Medical Center, 55 Lake Ave. North, Worcester, MA 01655, USA.
Ronghua
R
Zhuge
Kevin E
KE
Fogarty
Stephen P
SP
Baker
John G
JG
McCarron
Richard A
RA
Tuft
Lawrence M
LM
Lifshitz
John V
JV
Walsh
D000818:Animals; D002024:Bufo marinus; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008856:Microscopy, Fluorescence / Q000379:methods*; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D018408:Patch-Clamp Techniques; D024681:Potassium Channels, Calcium-Activated / Q000502:physiology*; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology*; D012519:Sarcoplasmic Reticulum / Q000502:physiology
D051036:Large-Conductance Calcium-Activated Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium