Store-operated Ca(2+) channels in human glomerular mesangial cells.
Experiments were performed to identify the biophysical properties of store-operated Ca(2+) channels (SOC) in cultured human glomerular mesangial cells (MC). A fluorometric technique (fura 2) was utilized to monitor the change in intracellular calcium concentration ([Ca(2+)](i)) evoked by elevating external [Ca(2+)] from 10 nM to 1 mM (Delta[Ca(2+)]). Under control conditions, Delta[Ca(2+)] averaged 6 nM and was unaffected by elevating bath [K(+)]. After treatment with 1 microM thapsigargin to deplete the intracellular Ca(2+) store, the change in [Ca(2+)](i) (Delta[Ca(2+)](th)) averaged 147 +/- 16 nM. In thapsigargin-treated MC studied under depolarizing conditions (75 mM bath K(+)), Delta[Ca(2+)](th) was 45 +/- 7 nM. The Delta[Ca(2+)](th) response of thapsigargin-treated cells was inhibited by La(3+) (IC(50) = 335 nM) but was unaffected by 5 microM Cd(2+). In patch clamp studies, inward currents were observed in cell-attached patches with either 90 mM Ba(2+) or Ca(2+) in the pipette and 140 mM KCl in the bathing solution. The single-channel conductance was 2.1 pS with Ba(2+) and 0.7 pS with Ca(2+). The estimated selectivities were Ca(2+) > Ba(2+) >> K(+). These channels were sensitive to 2 microM La(3+), insensitive to 5 microM Cd(2+), and voltage independent, with an average channel activity (NP(o)) of 1.02 at command potential (-V(p)) ranging from 0 to -80 mV. In summary, MC exhibited an electrogenic Ca(2+) influx pathway that is suggestive of Ca(2+) entry through SOC, as well as a small-conductance divalent-selective channel displaying biophysical properties consistent with SOC. Based on estimates of whole cell Ca(2+) influx derived from our data, we conclude that SOC with low single-channel conductance must be highly abundant in MC to allow significant capacitative Ca(2+) entry in response to depletion of the intracellular store.
Ma
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Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha 68198-4575, USA.
Smith
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S
Child
A
A
Carmines
P K
PK
Sansom
S C
SC
D055592:Biophysical Phenomena; D001703:Biophysics; D002104:Cadmium / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016257:Fura-2; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007811:Lanthanum / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D019284:Thapsigargin / Q000494:pharmacology
D015220:Calcium Channels; D004791:Enzyme Inhibitors; D002104:Cadmium; D019284:Thapsigargin; D007811:Lanthanum; D000252:Calcium-Transporting ATPases; D016257:Fura-2