Stretch-inactivated ion channels coexist with stretch-activated ion channels.

Stretch-activated ion channels of animal, plant, bacterial, and fungal cells are implicated in mechanotransduction and osmoregulation. A new class of channel has now been described that is stretch-inactivated. These channels occur in neurons, where they coexist with stretch-activated channels. Both channels are potassium selective. The differing stretch sensitivities of the two channels minimize potassium conductance over an intermediate range of tension, with the consequence that, over this same range, voltage-gated calcium channels are most readily opened. Thus, by setting the relation between membrane tension and transmembrane calcium fluxes, stretch-sensitive potassium channels may participate in the control of calcium-dependent motility in differentiating, regenerating, or migrating neurons.

Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL.

MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (Po) has a steep sigmoidal dependence on T, with a midpoint (T1/2) of 11.8 dyn/cm. The maximal slope sensitivity of Po/Pc was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was DeltaE = 18.6 kBT. If the mechanosensitivity arises from tension acting on a change of in-plane area (DeltaA), the free energy, TDeltaA, would correspond to DeltaA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest DeltaA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to DeltaA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.

Stretch-activated ion channels in growth cones of snail neurons.

Using single-channel recording, we show that neurons contain ion channels sensitive to membrane tension. Neurons isolated from the snail, Lymnaea stagnalis, actively rearborized in culture yielding cell bodies and growth cones suitable for patch clamping. All neurons contained, in both their soma and growth cones (at a density of approximately 1-2 micron-2), stretch-activated channels highly selective for K+. The presence of this mechanosensitive channel in the motile region of the neuron, a region characterized by insertion of new membrane--the growth cone--is of particular interest. Under physiological conditions, the channel was permeable to K+, but not to Na+ or Cl-. Its conductance to K+ under these conditions was approximately 44 pS. Channel activation was steeply dependent on membrane tension, showing thresholds at between -50 to -100 mm Hg (suction was applied through the recording pipette). Kinetic analysis indicated that the stretch-dependent increase in the channel's open probability was related to a long closed state rather than to one of the open states. Given the importance of Ca2+ in the regulation of growth cone motility, we speculate that this stretch-activated K+ channel could play a role in neurite elongation by a tension-dependent modulation of membrane voltage which in turn would act on voltage-gated Ca2+ channels.

Mechanically induced calcium mobilization in cultured endothelial cells is dependent on actin and phospholipase.

We sought to evaluate the mechanisms by which mechanical perturbation elevates intracellular calcium in endothelial cells. We report that the transient elevation in intracellular calcium in cultured bovine aortic endothelial cells (BAEC) in response to gentle perturbation with the side of a micropipette was not blocked by depolarization (external K+, 130 mmol/L), nifedipine (10 mumol/L), or Bay K 8644 R(+) (10 mumol/L). Thus, voltage-dependent calcium channels were not involved in the response. Also, amiloride (10 mumol/L) and tetraethylammonium (1 mmol/L) had no effect on calcium mobilization, indicating that Na+ and K+ transporters were not involved. Pretreatment of the cells with the phospholipase C and phospholipase A2 inhibitor manoalide (10 mumol/L) for 10 minutes at 37 degrees C completely abolished the calcium response, as did a 10-minute pretreatment with the inhibitor of actin polymerization, cytochalasin B (1 mumol/L). We observed an inhibitory effect of the phospholipase A2 and phospholipase C inhibitor 4-bromophenacyl bromide (10 mumol/L) on the mechanical response of BAEC that was not as potent as that observed with manoalide. To examine the role of arachidonic acid (AA) and subsequent metabolites that may be released after a putatively mechanical activation of phospholipase A2, we exposed BAEC to exogenous AA. We found that continued exposure of BAEC for 5 minutes to 10 nmol/L to 10 mumol/L AA caused no elevation of intracellular calcium. If mechanical stimulation activates phospholipase A2, the liberated AA and subsequent metabolites do not appear to have much effect on BAEC intracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanical perturbation of cultured human endothelial cells causes rapid increases of intracellular calcium.

In first-passage human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (passages 13-16), exposure to gentle mechanical perturbation using a micropipette caused a transient rise in intracellular calcium concentration ([Ca2+]i). The increase in calcium concentration ([Ca2+]) occurred each time the cell was nudged. Three responses were evoked in each of 27 cells using 5 independent HUVEC harvests. Increase in [Ca2+] returned to near baseline levels within approximately 30 s. The stimulus did not cause membrane puncture, as indicated by 1) absence of rapid dye leakage, 2) regulated nature of the [Ca2+] response, 3) absence of membrane blebbing, and 4) repeatable nature of the response in the same cell. As an alternative stimulus, we created very narrow fluid streams (1- to 2-microns diam) from a pressurized pipette that generated shear stresses of approximately 0.001-0.1 dyn/cm2 on the cells. However, these low-shear streams had little effect on [Ca2+]i. The poke-induced change in [Ca2+] was not blocked by lowering extracellular [Ca2+] ([Ca2+]o; 10 microM). In the absence of [Ca2+]o, however, HUVEC did not respond to the first poke, indicating a requirement for some [Ca2+]o as a mediator of signaling. After several poke-induced responses, [Ca2+]i could still be released by caffeine (100 microM), indicating the integrity of the intracellular release mechanism(s). These studies indicate that the response of an endothelial cell to a membrane-deforming event involves a priming step utilizing [Ca2+]o, which facilitates the transient increase of [Ca2+]i.

A hot aldehyde-peroxide fixation method for electron microscopy of the free-living nematode Caenorhabditis elegans.

An improved method for the fixation of the third and fourth larval stages and adults of Caenorhabditis elegans has been developed. Worms are placed in a mixture of 1.5% paraformaldehyde and 1.0% glutaraldehyde at pH 7.0 and 70 C and the suspension promptly cooled in a water bath at 20 C for 1 hr. The fixed worms are then immersed in a mixture of 5% glutaraldehyde and hydrogen peroxide at 4 C for 1 hr, stained en bloc in uranyl acetate, and embedded in resin for electron microscopy. The procedure results in superior fixation, particularly of microfilaments and microtubules. The high temperature of the initial fixation straightens the worms and thus facilitates serial sectioning.

Activation by curare of acetylcholine receptor channels in a murine skeletal muscle cell line.

Curare action on nicotinic acetylcholine receptors has a number of facets, of which the best known is competitive antagonism. Here we describe the weak agonist action of 10(-5) M curare on the murine skeletal muscle cell line, G8. Although curare induces no depolarization in G8 cells, single-channel recordings reveal short-lived curare-induced currents. A feature of these brief events is the multiplicity of conductance levels (of the four levels with conductances of 48, 37, 14, and 6 pS, none had a lifetime greater than 1.5 ms). Most well-resolved events (about 17% of which are to a subconductance) last less than 0.5 ms, with activation occurring predominantly as isolated events rather than in bursts. Agonism is not, however, a high probability action for curare: calculations based on the frequency of events at half-saturating conditions suggest that curare-induced channel openings occur during less than 1% of acetylcholine receptor-curare binding episodes. The outcome is (a) an agonist action too feeble to perturb the membrane voltage and (b) a powerful competitive antagonist action.

Ca2+ uptake in GH3 cells during hypotonic swelling: the sensory role of stretch-activated ion channels.

Hypotonic cell swelling triggers an increase in intracellular Ca2+ concentration that is deemed responsible for the subsequent regulated volume decrease in many cells. To understand the mechanisms underlying this increase, we have studied the Ca2+ sources that contribute to hypotonic cell swelling-induced Ca2+ increase (HICI) in GH3 cells. Fura 2 fluorescence of cell populations revealed that extracellular, but not intracellular, stores of Ca2+ were required. HICI was abolished by nifedipine, a blocker of L-type Ca2+ channels, and Gd3+, a nonspecific blocker of stretch-activated channels (SACs), suggesting two components for the Ca2+ membrane pathway: L-type Ca2+ channels and SACs. Using HICI as an assay, we found that venom from the spider Grammostola spatulata could block HICI without blocking L-type Ca2+ channels. The venom did, however, block SAC activity. This suggests that Ca(2+)-permeable SACs, rather than L-type Ca2+ channels, are the sensing elements for HICI. These results support the model for volume regulation in which SACs, activated by an increase of the membrane tension during hypotonic cell swelling, trigger HICI, leading to a volume decrease.