Abstract
We examined the single channel properties and regulation of store-operated calcium channels (SOCC). In human submandibular gland cells, carbachol (CCh) induced flickery channel activity while thapsigargin (Tg) induced burst-like activity, with relatively lower open probability (NP(o)) and longer mean open time. Tg- and CCh-activated channels were permeable to Na(+) and Ba(2+), but not to NMDG, in the absence of Ca(2+). The channels exhibited similar Ca(2+), Na(+), and Ba(2+) conductances and were inhibited by 2-aminoethoxydiphenylborate, xestospongin C, Gd(3+), and La(3+). CCh stimulated flickery activity changed to burst-like activity by (i) addition of Tg, (ii) using Na(+) instead of Ca(2+), (iii) using Ca(2+)-free bath solution, or (iv) buffering [Ca(2+)](i) with BAPTA-AM. Buffering [Ca(2+)](i) induced a 2-fold increase in NP(o) of Tg-stimulated SOCC. Reducing free [Ca(2+)] in the endoplasmic reticulum with the divalent cation chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), induced burst-like channel activity similar to that seen with CCh + Tg. Thus, SOCC is activated by stimulation of muscarinic receptors, inhibition of the sarcoendoplasmic Ca(2+) pump, and lowering [Ca(2+)] in the internal store. Importantly, SOCC activity depends on [Ca(2+)](i) and the free [Ca(2+)] in the internal store. These novel findings reveal that SERCA plays a major role in the gating of SOCC by (i) refilling the internal Ca(2+) store(s) and (ii) decreasing the [Ca(2+)](i)-dependent inhibition.
Highlights
Calcium influx into non-excitable cells is mediated via storeoperated Ca2ϩ entry channel(s) (SOCC),1 which are activated by the depletion of Ca2ϩ from intracellular calcium store(s) [1,2,3]
We have examined the single channel characteristics of SOCC activated by the muscarinic agonist, carbachol, the SERCA inhibitor, thapsigargin, and the permeant divalent cation chelator, TPEN, in the human submandibular gland cell line (HSG) by using the cell-attached patch clamp technique
This study describes the characteristics and regulation of an endogenous SOCC in HSG cells
Summary
Three main mechanisms currently proposed for the activation of SOCC include involvement of a diffusible messenger, recruitment of intracellular vesicles, and a physical interaction between the SOCC and IP3R [4, 5] According to the latter, i.e. the conformational coupling model [2], the IP3R acts as the sensor of the internal Ca2ϩ store and conveys the signal to the SOCC via a conformational change that results in activating the plasma membrane channel. Studies with the Trp family of putative Ca2ϩ channel proteins, which have been proposed as molecular components of the SOCC, have provided information regarding the possible involvement of IP3R and PIP2 hydrolysis in storeoperated Ca2ϩ influx [9, 10]. We show that the gating of this channel is regulated by the function of SERCA, which determines the [Ca2ϩ] in the internal Ca2ϩ store and in the vicinity of SOCC
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