A Ca2+-permeable non-selective cation channel activated by depletion of internal Ca2+ stores in single rabbit portal vein myocytes - PubMed (original) (raw)

A Ca2+-permeable non-selective cation channel activated by depletion of internal Ca2+ stores in single rabbit portal vein myocytes

A P Albert et al. J Physiol. 2002.

Abstract

In vascular smooth muscle cells many agonists cause the release of Ca2+ ions from internal stores. An important problem concerns the mechanism by which the intracellular stores are refilled subsequent to depletion. In the present study, we describe the properties of a Ca2+-permeable non-selective cation channel current that is activated in rabbit portal vein myocytes by depletion of internal Ca2+ stores. Application of cyclopiazonic acid (CPA), which depletes internal Ca2+ stores, activated whole-cell currents that had a reversal potential (E(r)) of about +50 mV in 1.5 mM external Ca2+ (Ca2+o). In 0 mM Ca2+o, the currents were larger and E(r) was approximately 0 mV. Application of CPA and caffeine during cell-attached recording activated single inward channel currents at negative potentials, which had a slope conductance of 2-3 pS and an E(r) of +20 mV. The slope conductance in 0 and 110 mM Ca2+o was 7 and 1.5 pS, respectively, and E(r) values indicated that these non-selective cation channels are highly permeable to Ca2+ ions. Bath application of the cell-permeant Ca2+ chelator, BAPTA-AM, also activated similar currents, indicating that these channels are not activated by Ca2+. Spontaneous channel currents with similar properties to store-operated channels were observed in some patches. Application of W-7, an inhibitor of the Ca2+-binding protein calmodulin, also activated similar Ca2+-permeable channel currents. In conclusion, it is demonstrated that agents that deplete Ca2+ stores and inhibit calmodulin binding activate Ca2+-permeable non-selective cation channel currents in rabbit portal vein myocytes. These channels may have an important role in vascular smooth muscle in providing an influx of Ca2+ to refill depleted internal Ca2+ stores and appear to possess different characteristics to store-operated channels described in other vascular smooth muscle preparations.

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Figures

Figure 1

Figure 1. CPA activates a non-selective cation current in rabbit portal vein smooth muscle cells recorded with the whole-cell method

A, the mean time course of the CPA-evoked cation currents recorded at −50 mV in either 1.5 m

m

(•) or 0 m

m

(○) Cao2+. Each data point is the mean of at least 4 cells. B, examples of I_–_V relationship of the CPA-evoked cation currents recorded in either 1.5 m

m

or 0 Cao2+. Note the larger CPA-evoked current, linear I_–_V relationship and more negative reversal potential (_E_r) in 0 m

m

Cao2+ compared to 1.5 m

m

Cao2+. C, the mean I-V relationships of the CPA-evoked cation currents in 1.5 m

m

(•) or 0 m

m

(○) Cao2+. Each point is the mean of 5 cells. The plots have been normalized to the amplitudes of the whole-cell cation currents in 1.5 m

m

Cao2+ at −50 mV (= −1 on _y_-axis).

Figure 2

Figure 2. Application of CPA or caffeine activates single channel currents in cell-attached patches from rabbit portal vein smooth muscle cells

A, using 126 m

m

NaCl with 1.5 m

m

Ca2+ patch pipette solution, bath application of 10 μ

m

CPA activated single channel currents in a patch that had not previously shown single channel activity. Note that inward currents are represented as downward deflections and that there is no CPA-evoked single channel activity at +100 mV. Continuous lines indicate the closed level, whereas dashed lines indicate open levels. B, the I_–_V relationship of the CPA-evoked single inward channel currents shown in A had a slope conductance (γ) of 2.1 pS between −120 and −40 mV and an extrapolated _E_r (↓) of +23 mV. C, bath application of 10 m

m

caffeine evoked single channel currents in a different cell-attached patch. Note the multiple single channel current openings at negative patch potentials. D, the I_–_V relationship of the caffeine-evoked single inward currents shown in C that had a slope conductance of 2 pS between −120 and −40 mV and an extrapolated _E_r (↓) of +22 mV.

Figure 3

Figure 3. Application of the cell-permeant Ca2+ chelator, BAPTA-AM, activates inward channel currents that have similar properties to the CPA- and caffeine-evoked channel currents

A, activation of inward channel currents at negative membrane potentials after bath application of BAPTA-AM for ∼20 min. Note that no channel currents could be observed at positive membrane potentials. B, the I_–_V relationship of the channel currents shown in A. The channel currents had a slope conductance of 2.3 pS between −120 and −40 mV and an extrapolated _E_r (↓) of +25 mV.

Figure 4

Figure 4. Application of CPA activates single cation channels that are permeable to Ca2+ ions

A, with a 110 m

m

CaCl2 patch pipette solution, bath application of 10 μ

m

CPA activated single cation currents in a previously quiescent patch. The CPA-evoked single cation currents were only observed at negative patch potentials. B, the pooled I_–_V relationships of CPA-evoked single cation currents recorded with 110 m

m

CaCl2 patch pipette solution. The I_–_V relationship had a slope conductance of 1.5 pS between −120 and −40 mV and an extrapolated _E_r (↓)of +86 mV. Each point is the mean of at least 4 patches.

Figure 5

Figure 5. Cell-attached patches contain spontaneous single channel currents with characteristics similar to the CPA- and caffeine-evoked cation channels

A, spontaneous channel activity between −40 and −120 mV in one patch recorded with 126 m

m

NaCl patch pipette solution. B, pooled I_–_V relationship of spontaneous non-selective cation channels recorded with 126 m

m

NaCl with 1.5 m

m

Cao2+ (•) or 110 m

m

CaCl2 (○) in the patch pipette solution. In 126 m

m

NaCl, the I_–_V relationship had a slope conductance of 2 pS between −120 and −40 mV and an extrapolated _E_r (↓) of +18 mV. In 110 m

m

CaCl2, the I_–_V relationship had a slope conductance of 1.3 pS and an extrapolated _E_r (↓) of + 98 mV. Each point is the mean of at least 4 patches.

Figure 6

Figure 6. Open lifetime distributions of the CPA-, caffeine-, BAPTA-AM-evoked and spontaneous cation channel currents recorded with a 126 mm NaCl patch pipette solution

The open lifetime distributions of CPA- (A), caffeine- (B), BAPTA-AM-evoked (C) and spontaneous (D) cation channels could all be fitted with (continuous lines) the sum of two exponentials with time constants of approximately 5 ms (Oτ1) and 30 ms (Oτ2). The holding potential was −80 mV in all cases.

Figure 7

Figure 7. Characteristics of non-selective cation channel currents in the absence of Cao2+

A, spontaneous channel currents recorded at different membrane potentials with a 0 m

m

Cao2+ patch pipette solution. Note that outward channel currents (denoted by upward deflections) can be observed at positive membrane potentials. B, the I_–_V relationship of the channel currents shown in A. The I_–_V relationship was linear between −110 and +80 mV with a slope conductance of 8 pS and an _E_r of −4 mV. C, open time distribution of the channel currents shown in A at −110 mV. The open times could be described by the sum of two exponentials with time constants of 5.4 ms (Oτ1) and 32 ms (Oτ2).

Figure 8

Figure 8. Application of the calmodulin inhibitor, W-7, activates single channel currents with properties that are similar to CPA-evoked cation channel currents

A, with a 126 m

m

NaCl pipette solution, bath application of 50 μ

m

W-7 evoked single channel currents at negative patch potentials. B, the I_–_V relationship of the W-7-evoked single channels currents shown in A. C, at −80 mV, the open time distribution of the W-7-evoked single channels shown in A could be described by the sum of two exponentials with time constants of 5 ms (Oτ1) and 36 ms (Oτ2).

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References

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