Using the inside-out patch-clamp technique, large-conductance Ca2+ -activated K+ channel (BK(Ca)) currents were recorded from coronary artery smooth muscle cells. Cytochalasin D, an actin filament disrupter, increased channel activity ( NP(o), where N is the number of channels and P(o) the open probability), and this increase was reversed by phalloidin, an actin filament stabilizer. NP(o) was also increased by colchicine, a microtubule disrupter, and decreased by taxol, a microtubule stabilizer. With the stepwise increase of negative pressure in the patch pipettes, the activity of BK(Ca) gradually increased: the maximum effect (527% increase in NP(o)) was achieved at -40 cmH(2)O and the half-maximum effect at -25 cmH(2)O. The increase in NP(o) in response to negative pressure was abolished by phalloidin but not by taxol. These results imply that both actin filaments and microtubules inhibit the opening of BK(Ca) in coronary artery smooth muscle cells, but that only actin filaments are involved in the stretch sensitivity of BK(Ca).
The cells of vertebrates exposed to hyposmotic media, initially swell by osmotic water equilibration, but subsequently regulate their volume (regulatory volume decrease, RVD) by a loss of KCl and water (for review, see references [1][2][3]). The mechanisms of osmoregulatory K ϩ and Cl Ϫ efflux have not been completely understood. However, K ϩ and Cl Ϫ channels have been proposed to play an important role in RVD in various kinds of cells, such as those in frog urinary bladder [4], human lymphocytic cells [5], human epithelial cells [6], human platelets [7], and epithelial cells of frog skin [8].Both extracellular and intracellular [Ca 2ϩ ] are too low in animals to be useful as osmolytes. However, the Ca 2ϩ role during RVD has been recognized for many years. Many patch-clamp studies have found that the activation of volume-regulatory K ϩ channels is controlled by Ca 2ϩ (for review, see references [9,10]). Some experiments indicated that I K(Ca) was activated by an influx of Ca 2ϩ when the cell was superfused with hyposmotic solution [6,11,12]. Other experiments have shown that I K(Ca) is activated by an intracellular Ca 2ϩ signal that is released from intracellular calcium stores during RVD [13]. But the effect of hyposmotic swelling on I K(Ca) and the role of intracellular or extracellular Ca 2ϩ in gastric smooth muscle cells has not been investigated.In our previous study, we reported that the voltageoperated calcium current (I Ca ) was increased [14] and that the volume-sensitive chloride current (I Cl ) was activated by hyposmotic swelling [15] in gastric antral myocytes of the guinea-pig. In the present study, we Key words: gastric smooth muscle cell, Ca 2ϩ -activated K ϩ current, Ca 2ϩ -induced Ca 2ϩ release, swelling.Abstract: In our study of the effects of hyposmotic swelling on the Ca 2ϩ -activated potassium currents [I K(Ca) ] and its mechanism, we employed the whole-cell patch clamp technique using the gastric antral circular myocytes of the guinea-pig. Hyposmotic swelling efficiently increased I K(Ca) , and the extent of changes in I K(Ca) was sharply dependent on the osmolarity of the perfusion solutions. When the calcium-free solution (EGTA 10 M added in calcium-free solution) was superfused, I K(Ca) was not increased by the hyposmotic swelling. Gadolinium (Gd 3ϩ ) 100 nM, a blocker of the stretch-activated nonselective cation channel, blocked the activation of I K(Ca) induced by hyposmotic swelling, but nicardipine 5 M (the Ltype calcium channel blocker) did not. Heparin 3 mg/ml, a potent inhibitor of inositol triphosphate receptor (InsP 3 R), did not inhibit the response, and caffeine 1 mM (the agonist for calcium-induced calcium release [CICR]) imitated the effect of hyposmotic swelling. Ryanodine (15 M), markedly inhibited the effect. These results suggest that hyposmotic swelling activates I K(Ca) , and the activation is associated with CICR, which is triggered by extracellular calcium influx through the stretch-activated channel (SA channel).
We investigated the mechanosensitivity of voltage-gated K+ channel (VGPC) currents by using whole-cell patch clamp recording in rat trigeminal ganglion (TG) neurons. On the basis of biophysical and pharmacological properties, two types of VGPC currents were isolated. One was transient (I(K,A)), the other sustained (I(K,V)). Hypotonic stimulation (200 mOsm) markedly increased both I(K,A) and I(K,V) without affecting their activation and inactivation kinetics. Gadolinium, a well-known blocker of mechanosensitive channels, failed to block the enhancement of I(K,A) and I(K,V) induced by hypotonic stimulation. During hypotonic stimulation, cytochalasin D, an actin-based cytoskeletal disruptor, further increased I(K,A) and I(K,V), whereas phalloidin, an actin-based cytoskeletal stabilizer, reduced I(K,A) and I(K,V). Confocal imaging with Texas red-phalloidin showed that actin-based cytoskeleton was disrupted by hypotonic stimulation, which was similar to the effect of cytochalasin D. Our results suggest that both I(K,A) and I(K,V) are mechanosensitive and that actin-based cytoskeleton is likely to regulate the mechanosensitivity of VGPC currents in TG neurons.
Hyposmotic membrane stretch potentiated muscarinic receptor agonist-induced depolarization of membrane potential, which is related to hyposmotic membrane stretch-induced increase of muscarinic current.
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