Gating of store-operated channels by conformational coupling to ryanodine receptors.

We report here that RyRs interact with and gate the store-operated hTrp3 and Icrac channels. This gating contributes to activation of hTrp3 and Icrac by agonists. Coupling of hTrp3 to IP3Rs or RyRs in the same cells was found to be mutually exclusive. Biochemical and functional evidence suggest that mutually exclusive coupling reflects clustering and segregation of hTrp3-IP3R and hTrp3-RyR complexes in plasma membrane microdomains. Gating of CCE by RyRs indicates that gating by conformational coupling is not unique to skeletal muscle but is a general mechanism for communication between events in the plasma and endoplasmic reticulum membranes.

Miniature Ca2+ channels in excised plasma-membrane patches: activation by IP3.

In the present work, we characterized the receptor properties and the conductive features of the inositol (1,4,5)-trisphosphate (IP3)-activated Ca2+ channels present in excised plasma-membrane patches obtained from mouse macrophages and A431 cells. We found that the receptor properties of the channels tested were similar to those of the IP3 receptor (IP3R) expressed in the endoplasmic reticulum (ER) membrane. These properties include activation by IP3, inhibition by heparin, time-dependent inactivation by high IP3 concentrations, activation by guanosine 5'o-thiotriphosphate and regulation by arachidonic acid. On the other hand, in terms of conductive properties, the channel closely resembles Ca2+-release-activated Ca2+ channels (Icrac). These conductive properties include extremely low conductance (approximately 1 pS), very high selectivity for Ca2+ over K+ (PCa/PK>1000), inactivation by high intracellular Ca2+ concentration and, importantly, strong inward rectification. Notably, the same channel was activated by: (1) agonists in the cell-attached mode of channel recording, and (2) cytosolic IP3 after patch excision. Although the possibility cannot be completely excluded that a novel type of IP3R is expressed exclusively in the plasma membrane, in their entirety our findings suggest that the plasma membrane of mouse macrophages and A431 cells contains Icrac-like Ca2+ channels coupled to an IP3-responsive protein which displays properties similar to those of the IP3R expressed in the ER membrane.

Involvement of Ca2+-induced Ca2+ release in the biphasic Ca2+ response evoked by readdition of Ca2+ to the medium after UTP-induced store depletion in A431 cells.

We have recently shown that the Ca2+ response in endothelial cells evoked by readdition of Ca2+ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca2+ release from Ca2+ stores sensitive to both inositol 1,4, 5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca2+]i. We found that the amplitude of the Ca2+ response evoked by Ca2+ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca2+ release, but did positively correlate with the initial [Ca2+]i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca2+ responses) revealed that there is an accelerating rise in [Ca2+]i during the biphasic response. Application of ryanodine during the plateau-shaped Ca2+ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca2+ release, but it caused a further [Ca2+]i rise when [Ca2+]i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca2+ after agonist-evoked store depletion can evoke Ca2+-induced Ca2+ release. This indicates that Ca2+ entry may be overestimated by this widely used protocol.

Low-conductance high selective inositol (1,4,5)-trisphosphate activated Ca2+ channels in plasma membrane of A431 carcinoma cells.

In many cells, activation of receptors coupled to PIP2 turnover results in Ca2+ release from the intracellular stores accompanied by Ca2+ influx across the PM. It is not well established yet whether Ca2+ influx is activated by IP3 or by an unknown signal generated upon Ca2+ store depletion. We report here a single-channel study of low-conductance IP3-activated channels of very high selectivity for Ca2+ in the PM of A431 carcinoma cells. The channels are strongly potential dependent and sensitive to [Ca2+]i within the physiological range. The data obtained argues for IP3 acting directly on plasma membrane Ca2+ channels.

Effect of intracellular calcium on ATP-activated, GTP-dependent calcium channels in rat macrophages.

1. In order to study the effect of intracellular free Ca2+ concentration ([Ca2+]i) on the activity of ATP-activated, GTP-dependent Ca2+ channels in rat macrophages, experiments were performed using the inside-out configuration of the patch-clamp technique. 2. Channel activity was observed in the cell-attached mode when 100 microM ATP was added to the pipette solution containing 105 mM Ba2+, but it disappeared rapidly after patch excision. The activity could be restored by the application of 100 microM GTP or GTP gamma S onto the internal surface of the plasma membrane. 3. The properties of the GTP gamma S-evoked channels are identical to those of channels activated by extracellular application of ATP. The channels exhibited four current sublevels with conductances of about 3.5, 7, 10 and 15 pS when 105 mM Ba2+ was the only permeant cation. The extrapolated reversal potentials were similar for all the sublevels and averaged about +40 mV. 4. Elevation of [Ca2+]i within the range 0.01-1 microM resulted in a decrease in mean inward current. The half-maximal value of the mean current was about 0.08 microM. 5. This decreases in mean current resulted from a redistribution of sublevel occupancies: the 1st sublevel tended to be come more abundant with elevation of [Ca2+]i, while the relative weights of the high-conductance 3rd and 4th sublevels decreased. 6. The open-channel current fell with an increase in [Ca2+]i as quickly as the mean current did, indicating that the sublevel redistribution alone is sufficient to produce the revealed decrease in net inward current. 7. It is concluded that [Ca2+]i elevation does not fix the channel in a closed state but rather decreases the ability of the channel to operate in high-conductance states.

ATP-activated inward current and calcium-permeable channels in rat macrophage plasma membranes.

1. To study mechanisms of receptor-operated Ca2+ influx in non-excitable cells, membrane currents of rat peritoneal macrophages were recorded using whole-cell cell-attached and outside-out configurations of the patch clamp technique. Under whole-cell recording conditions, ATP applied in micromolar concentrations elicited an inward current response when the bath solution contained Ba2+, Ca2+ or Na+ as the only permeant cations. 2. Increasing the Mg2+ concentration had an inhibitory effect on the ATP-induced inward current indicating that the active form of ATP responsible for the cation entry is ATP4-. The nucleotide potency order was ATP > ATP gamma S > ADP. UTP was completely ineffective (n = 19). The data obtained are consistent with the ATP receptor being of the P2Z type. 3. The macrophage plasma membrane was impermeable to Tris+ during the ATP-induced current at ATP4- concentrations varying from 0.07 to 500 microM. At higher concentrations, ATP produced a large inward steady-state current, which could be attributed to membrane permeabilization. 4. Activity of single channels was recorded when ATP was applied to the external surface of the patch membrane both in cell-attached and outside-out experiments. A specific property of the channels appeared to be the existence of at least four conductance sublevels. With 105 mM Ba2+ as the permeant cation, the conductance sublevels were 3.5, 7, 10 and 15 pS. With 10 mM Ca2+ the sublevel conductances were equal to 4, 9, 13 and 17 pS. 5. The unitary conductance estimated from the whole-cell current noise analysis (3.5-4.5 pS for 105 mM Ba2+) was significantly lower than that obtained from single channel measurements at the main (3rd) current level, but it was very close to the conductance of the minimum (1st) level. 6. Extrapolated reversal potential values estimated from current-voltage curves for predominant conductance levels were equal to +40 and +26 mV for 105 mM Ba2+ and 10 mM Ca2+, respectively. The permeability ratios fell in the sequence: PCa:PBa:PK = 71.:29:1. Thus, ATP-activated channels in the macrophage membrane are rather selective for divalent vs. monovalent cations, with the predominant permeability being for Ca2+.

ATP-operated calcium-permeable channels activated via a guanine nucleotide-dependent mechanism in rat macrophages.

1. To elucidate the possible involvement of a G protein in ATP-evoked Ca(2+)-permeable channel activity, membrane currents of rat peritoneal macrophages were recorded using inside-out and cell-attached configurations of the patch clamp technique. 2. In inside-out experiments with a pipette solution containing 105 mM Ba2+, application of 100 microM GTP or GTP gamma S to the internal surface of the membrane elicited a rise in channel activity. This effect was observed in 49% of the patches investigated (n = 69). The mean value of NPo (N, number of open channels; Po, channel open probability) was equal to 0.49 +/- 0.27 (mean +/- S.E.M.; n = 16). The delay in the activity development was 21 +/- 8 s (n = 18) with 200 microM ATP added to the pipette solution and about 4 min (n = 5) without agonist in the pipette. Similar results were obtained with 10 mM Ca2+ as the only permeant cation. 3. Properties of GTP gamma S-evoked channels were identical to those of channels activated by extracellular application of ATP. The channels exhibited at least four conductance sublevels, the 4th one being the least frequent. With 105 mM Ba2+ as a permeant cation, sublevel conductances were 3.5, 7, 10 and 15 pS. Corresponding values for 10 mM Ca2+ were about 4, 9, 13 and 17 pS. Extrapolated reversal potential (Er) values were about +40 and +25 mV for Ba2+ and Ca2+, respectively. 4. The activity of channels with similar characteristics could be induced by the extracellular application of fluoride in cell-attached experiments without any agonist in the pipette solution.(ABSTRACT TRUNCATED AT 250 WORDS)