Local Ca(2+) transients and distribution of BK channels and ryanodine receptors in smooth muscle cells of guinea-pig vas deferens and urinary bladder.
1. The relationship between Ca(2+) sparks spontaneously occurring at rest and local Ca(2+) transients elicited by depolarization was analysed using two-dimensional confocal Ca(2+) images of single smooth muscle cells isolated from guinea-pig vas deferens and urinary bladder. The current activation by these Ca(2+) events was also recorded simultaneously under whole-cell voltage clamp. 2. Spontaneous transient outward currents (STOCs) and Ca(2+) sparks were simultaneously detected at -40 mV in approximately 50 % of myocytes of either type. Ca(2+) sparks and corresponding STOCs occurred repetitively in several discrete sites in the subplasmalemmal area. Large conductance Ca(2+)-dependent K(+) (BK) channel density in the plasmalemma near the Ca(2+) spark sites generating STOCs was calculated to be 21 channels microm(-2). 3. When myocytes were depolarized from -60 to 0 mV, several local Ca(2+) transients were elicited within 20 ms in exactly the same peripheral sites where sparks occurred at rest. The local Ca(2+) transients often lasted over 300 ms and spread into other areas. The appearance of local Ca(2+) transients occurred synchronously with the activation of Ca(2+)-dependent K(+) current (I(K,Ca)). 4. Immunofluorescence staining of the BK channel alpha-subunit (BKalpha) revealed a spot-like pattern on the plasmalemma, in contrast to the uniform staining of voltage-dependent Ca(2+) channel alpha1C subunits along the plasmalemma. Ryanodine receptor (RyR) immunostaining also suggested punctate localization predominantly in the periphery. Double staining of BKalpha and RyRs revealed spot-like co-localization on/beneath the plasmalemma. 5. Using pipettes of relatively low resistance, inside-out patches that included both clustered BK channels at a density of over 20 channels microm(-2) and functional Ca(2+) storage sites were obtained at a low probability of approximately 5%. The averaged BK channel density was 3-4 channels microm(-2) in both types of myocyte. 6. These results support the idea that a limited number of discrete sarcoplasmic reticulum (SR) fragments in the subplasmalemmal area play key roles in the control of BK channel activity in two ways: (i) by generating Ca(2+) sparks at rest to activate STOCs and (ii) by generating Ca(2+) transients presumably triggered by sparks during an action potential to activate a large I(K,Ca) and also induce a contraction. BK channels and RyRs may co-localize densely at the junctional areas of plasmalemma and SR fragments, where Ca(2+) sparks occur to elicit STOCs.
Ohi
Y
Y
Department of Molecular and Cellular Pharmacology, Faculty of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuhoku, Nagoya 467-8603, Japan.
Yamamura
H
H
Nagano
N
N
Ohya
S
S
Muraki
K
K
Watanabe
M
M
Imaizumi
Y
Y
D000814:Aniline Compounds; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D007150:Immunohistochemistry; D007700:Kinetics; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D018613:Microscopy, Confocal; D009130:Muscle, Smooth / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000032:analysis / Q000502:physiology*; D024681:Potassium Channels, Calcium-Activated; D019837:Ryanodine Receptor Calcium Release Channel / Q000032:analysis / Q000502:physiology*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D001743:Urinary Bladder / Q000166:cytology*; D014649:Vas Deferens / Q000166:cytology*; D014966:Xanthenes
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D019837:Ryanodine Receptor Calcium Release Channel; D014966:Xanthenes; C059715:Fluo-3; D002118:Calcium