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Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4575
Experiments were performed to identify the biophysical
properties of store-operated Ca2+ channels (SOC) in
cultured human glomerular mesangial cells (MC). A fluorometric
technique (fura 2) was utilized to monitor the change in intracellular
calcium concentration
([Ca2+]i) evoked by
elevating external [Ca2+] from 10 nM to 1 mM
(
[Ca2+]). Under control conditions,
[Ca2+] averaged 6 nM and was unaffected by
elevating bath [K+]. After treatment with 1 µM thapsigargin to deplete the intracellular Ca2+ store,
the change in [Ca2+]i
([Ca2+]th) averaged 147 ± 16 nM. In thapsigargin-treated MC studied under depolarizing conditions
(75 mM bath K+),
[Ca2+]th was 45 ± 7 nM. The
[Ca2+]th response of
thapsigargin-treated cells was inhibited by La3+
(IC50 = 335 nM) but was unaffected by 5 µM
Cd2+. In patch clamp studies, inward currents were observed
in cell-attached patches with either 90 mM Ba2+ or
Ca2+ in the pipette and 140 mM KCl in the bathing solution.
The single-channel conductance was 2.1 pS with Ba2+ and 0.7 pS with Ca2+. The estimated selectivities were
Ca2+ > Ba2+ >> K+. These
channels were sensitive to 2 µM La3+, insensitive to 5 µM Cd2+, and voltage independent, with an average channel
activity (NPo) of 1.02 at command
potential (
Vp) ranging from 0 to 80 mV. In summary, MC exhibited an electrogenic
Ca2+ influx pathway that is suggestive of Ca2+
entry through SOC, as well as a small-conductance divalent-selective channel displaying biophysical properties consistent with SOC. Based on
estimates of whole cell Ca2+ influx derived from our data,
we conclude that SOC with low single-channel conductance must be highly
abundant in MC to allow significant capacitative Ca2+ entry
in response to depletion of the intracellular store.
cadmium; lanthanum; fura 2 fluorescence; patch clamp; thapsigargin; angiotensin II
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