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Piezo channels transduce mechanical stimuli into electrical and chemical signals to powerfully influence development, tissue homeostasis, and regeneration. Studies on Piezo1 have largely focused on transduction of "outside-in" mechanical forces, and its response to internal, cell-generated forces remains poorly understood. Here, using measurements of endogenous Piezo1 activity and traction forces in native cellular conditions, we show that cellular traction forces generate spatially-restricted Piezo1-mediated Ca 2+ flickers in the absence of externally-applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their flicker activity is enriched near force-producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca 2+ flickers allow spatial segregation of mechanotransduction events, and that mobility allows Piezo1 channels to explore a large number of mechanical microdomains and thus respond to a greater diversity of mechanical cues., Competing Interests: Competing interestsThe authors declare no competing interests.

Poly(A)+ mRNAs from the cerebral cortex of aged (24 months) and young adult (3 months) rats were isolated and injected into Xenopus oocytes to express functional neurotransmitter receptors and voltage-operated channels. Electrophysiological recordings of induced membrane currents were used as a measure of the relative amounts of mRNA encoding different receptors and channels, and to study their functional properties. There were no large differences apparent between mRNAs from aged and adult rats, in marked contrast to the dramatic (1000-fold) changes in mRNA expression that occur during embryonic and postnatal development. The membrane currents induced by glutamate or acetylcholine (ACh) application were roughly one third smaller in oocytes injected with mRNA from aged cerebral cortex than in oocytes injected with mRNA from adult cerebral cortex, whereas currents induced by gamma-aminobutyric acid (GABA), kainate or serotonin (5-HT) application, and by activation of voltage-operated Na+ and Ca2+ channels were not significantly different. We did not observe any age-related differences in the properties of the receptors and channels studied.

The ontogenetic development of poly(A)+ mRNAs coding for receptors to several neurotransmitters (kainate, glutamate, acetylcholine, and serotonin) and voltage-operated channels (sodium and calcium) was studied by isolating total poly(A)+ mRNA from the brains of rats at various developmental stages and injecting it into Xenopus oocytes. The oocytes translated the foreign mRNA and incorporated functional receptor/ion channel complexes into the cell membrane. Thus, recording of induced membrane currents in voltage-clamped oocytes gave a measure of the relative amounts of the different messengers. Responses induced by kainate, glutamate, acetylcholine, and serotonin all increased with age and reached a maximum in oocytes injected with mRNA from adult cortex. Messenger RNAs for the earliest ages examined, Embryonic Days 15 and 18, expressed little or no response to kainate, glutamate, or acetylcholine, while 50-70% of the adult response was reached by Postnatal Day 10. In contrast, the serotonin-induced response was already comparatively large (16% of the adult level) in oocytes injected with mRNA from Embryonic Day 15 brain and increased postnatally to adult levels. The expression of voltage-dependent sodium and calcium channels was small in oocytes injected with mRNA from embryonic animals and increased postnatally to reach a maximum in oocytes injected with mRNA from adult animals.

Poly(A+) messenger RNA was extracted from the electric lobe and medulla of Torpedo and injected into oocytes of Xenopus laevis. The synthesis and processing of proteins coded by the injected messenger RNA led to the incorporation of voltage-activated channels in the oocyte membrane. A large, well maintained outward current was recorded from injected oocytes in response to depolarization. Non-injected oocytes did not show this current. The reversal potential of the current varied according to the Nernst equation with external potassium concentration, indicating that it was largely carried by potassium ions. The maintained potassium current was not reduced by manganese (5 mM) or lanthanum ions (0.1 mM). Tetraethylammonium and aminopyridines blocked the potassium current. The block produced by 3,4-diaminopyridine was enhanced by previous activation, but diminished by strong depolarization. The amplitude of the potassium current increased over the approximate voltage range -30 to +50 mV, but reduced at more positive potentials. The decline of the current during maintained depolarization became slower as the membrane potential was made more positive, and the rate of onset of the current became faster. Estimates from noise analysis indicated that the slow potassium current passes through channels with a mean lifetime of about 14 ms and conductance of 14 pS (at -10 mV and room temperature). Injection of the messenger RNA also induced the formation of fast sodium and potassium channels activated by voltage, and channels activated by kainate.

1. Membrane currents were recorded from voltage-clamped Xenopus oocytes in response to bath application of various divalent cations. 2. In oocytes from 93 of 160 frogs tested, Co2+ ions evoked slow, oscillatory membrane currents. Sensitivity to Co2+ varied greatly between oocytes from different frogs, but was relatively consistent for oocytes taken from the same ovary. Oocytes with high sensitivity had response thresholds of 5-10 microM, and gave currents greater than 1 microA to 1 mM-CoCl2. In contrast, oocytes from some frogs gave no oscillatory response even to 10 mM-CoCl2. With responsive oocytes, Cd2+, Ni2+, Zn2+, Mn2+ and Cr2+ ions (5 microM to 1 mM) also elicited oscillations, whereas Sr2+, Ba2+ and Ca2+ (0.1-10 mM) showed very little activity, and Mg2+ ions, none. 3. Responses to divalent cation were well preserved in defolliculated oocytes, indicating they were generated in the oocyte membrane itself, and were not dependent on the presence of enveloping follicular cells. 4. The oscillatory currents reversed around -20 mV (the chloride equilibrium potential) and rectified strongly at potentials more negative than about -60 mV. The oscillations were mimicked by intraoocyte injection of inositol 1,4,5-trisphosphate (IP3), were largely preserved after removal of external Ca2+, but were abolished following chelation of intracellular Ca2+ by EGTA. Intraoocyte injection of Co2+ ions failed to generate oscillatory currents. 5. Currents elicited by divalent cations resembled the oocyte's oscillatory responses to acetylcholine and a serum protein. However, the response to divalent cations was not blocked by atropine and furthermore, the relative sensitivities to these agonists varied independently between oocytes from different frogs. 6. We conclude that extracellular Cd2+, Ni2+, Zn2+, Co2+, Mn2+ and Cr2+ interact with the oocyte surface to raise cytosolic levels of inositol phosphates. This causes mobilization of intracellular Ca2+, in turn activating Ca2+-gated Cl- channels in the oocyte membrane. 7. In addition to the large oscillatory currents, divalent cations generated small (5-50 nA), smooth, maintained currents associated with decreases in membrane conductance. The size and ionic basis of these currents varied between oocytes from different frogs. 8. Zinc ions also elicited smooth currents, associated with an increase in membrane conductance, and carried predominantly by K+. This response was specific to Zn2+ and occurred independently of oscillatory Cl- currents. The K+ current was abolished by defolliculation, was potentiated by the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine,and showed facilitation with K+ currents generated by the adenylate cyclase activator forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)

Poly(A)+ mRNAs extracted from embryonic chicken brain and from adult rat brain were fractionated on sucrose density gradients. The fractions were subsequently injected into Xenopus oocytes where the mRNA was translated. The products were processed and incorporated into the oocyte membrane where they formed functional neurotransmitter receptors and voltage-operated channels. Different mRNA fractions induced the incorporation of different transmitter receptors and voltage-operated channels into the oocyte membrane. These experiments provide a useful step towards the understanding of the structure and function of neurotransmitter receptors and channels.

Depolarization of follicle-enclosed oocytes of Xenopus laevis obtained from some donors elicits, in addition to other responses, a fast transient outward current. After holding the membrane potential at -100 mV this response begins to be activated by depolarizations to around -30 mV, and increases progressively as the voltage is raised further. A striking characteristic is that the current recovers only slowly (several seconds) from inactivation following a depolarizing pulse. Because of its outward direction and insensitivity to removal of extracellular chloride or addition of tetrodotoxin, the current probably arises largely through a flux of potassium ions. The current was abolished after treatment of oocytes with collagenase to remove enveloping cells, and although it was blocked by barium and zinc ions, tetraethylammonium was relatively ineffective. In addition, the potassium current was unaffected by 5 mM manganese, suggesting that it does not arise as a consequence of an influx of calcium into the oocyte.

Sodium channels and receptors to serotonin and kainate were 'transplanted' from human brain into frog oocytes, by isolating messenger RNA from a fetal brain, and injecting it into Xenopus laevis oocytes. The mRNA was translated by the oocyte and induced the appearance of functional receptors and channels in its membrane. This approach renders drug- and voltage-operated channels of the human brain more amenable to detailed study.

The role of N-glycosylation on the expression of functional brain neurotransmitter receptors and voltage-operated channels was studied by injecting Xenopus oocytes with mRNA from rat brain or chick optic lobe, and culturing them in the presence or absence of tunicamycin, an inhibitor of asparagine linked glycosylation. Electrophysiological recordings were then made to assess the amounts of functional receptors and channels present in the oocyte membrane. The appearance of gamma-aminobutyric acid (GABA) receptors and voltage-activated Na+ channels was profoundly reduced. In contrast, the functional expression of kainate receptors, and voltage-activated K+ and Ca2+ channels was much less affected. Thus, it seems that kainate receptors, and K+ and Ca2+ channels can be expressed and function normally without being glycosylated. On the other hand, GABA receptors and Na+ channels may need to be N-glycosylated in order to function properly, or to ensure their correct insertion into the membrane.

1. Application of serum to Xenopus oocytes elicits an oscillatory chloride membrane current, which begins after a latency of several seconds or minutes, and is mediated through a phosphoinositide-calcium signalling pathway. We studied the characteristics and origin of this latency in voltage-clamped oocytes. 2. Bath application of low doses of serum evoked responses beginning after latencies of 1 min or more. The latency decreased with increasing dose and reached a minimal value of several seconds that did not decrease with further increases in serum concentration. Experiments to study this minimal latency were done by applying brief 'puffs' of serum and other agonists at high concentrations from a local extracellular pipette. 3. The mean latency of the response evoked by local serum application was about 7 s (at 22-24 degrees C), but individual responses showed a wide variation, from 2 s to over 20 s. Diffusion of serum from the pipette tip to the membrane did not contribute appreciably to this delay, since short (less than 100 ms) delays were obtained when KCl was applied in the same way. 4. Currents evoked by acetylcholine and serotonin, in oocytes induced to acquire muscarinic and serotonergic receptors following injection of brain messenger RNA, began following latencies similar to that of the serum response. 5. The response latency was shorter when serum was applied to the vegetal rather than the animal hemisphere of the oocyte, even though smaller currents were obtained. 6. The latency showed a slight dependence upon membrane potential, becoming shorter with depolarization. 7. Cooling to temperatures below about 22 degrees C produced a striking lengthening of the delay, corresponding to a Q10 of about 5. In contrast, above 22 degrees C the temperature dependence was slight, with a Q10 of about 1.25. 8. Intracellular injections of calcium and inositol 1,4,5-trisphosphate (IP3) evoked chloride currents with short (a few tens of milliseconds) latency. Short (100 ms) latency responses were also evoked when intracellularly loaded caged IP3 was photolysed by strong illumination, but weak illumination gave responses with latencies of over 1 min. 9. Measurements of intracellular free calcium, made with Fura-2 and Indo-1, showed an increase following serum application beginning coincident with the onset of the membrane current response.(ABSTRACT TRUNCATED AT 400 WORDS)

Poly(A)+ mRNA extracted from the electric organ of Torpedo was fractionated by sucrose density gradient centrifugation. After injection into Xenopus oocytes one mRNA fraction induced the appearance of chloride channels in the oocyte membrane. Many of these channels were normally open, and the ensuing chloride current kept the resting potential of injected oocytes close to the chloride equilibrium potential. When the membrane was hyperpolarized, the chloride current was reduced. A separate fraction of mRNA induced the incorporation of acetylcholine receptors into the oocyte membrane. When translated in a cell-free system this fraction directed the synthesis of the alpha, beta, gamma, and delta subunits of the acetylcholine receptor. In contrast, the mRNA fraction that induced the chloride channels caused the synthesis of the delta subunit, a very small amount of alpha, and no detectable beta or gamma subunits. This suggests that the size of the mRNA coding for the chloride channel is similar to the preponderant species of mRNA coding for the delta subunit of the acetylcholine receptor.

Xenopus oocytes were used to express polyadenylated messenger RNAs (mRNAs) encoding acetylcholine receptors and voltage-activated sodium channels from innervated and denervated skeletal muscles of cat and rat. Oocytes injected with mRNA from denervated muscle acquired high sensitivity to acetylcholine, whereas those injected with mRNA from innervated muscle showed virtually no response. Hence the amount of translationally active mRNA encoding acetylcholine receptors appears to be very low in normally innervated muscle, but increases greatly after denervation. Conversely, voltage-activated sodium currents induced by mRNA from innervated muscle were about three times larger than those from denervated muscle; this result suggests that innervated muscle contains more mRNA coding for sodium channels. The sodium current induced by mRNA from denervated muscle was relatively more resistant to block by tetrodotoxin. Thus a proportion of the sodium channels in denervated muscle may be encoded by mRNAs different from those encoding the normal channels.

1. Changes in end-plate channel properties resulting from substitution of Sr2+ for Ca2+ in the Ringer solution have been analysed at the voltage clamped frog end-plate, by recording m.e.p.c.s and ACh induced noise variance. 2. In 2 mM-Sr2+--Ringer the peak size of m.e.p.c.s showed a very small increase, and the time constant of the decay phase (tau m.e.p.c.), at any given voltage, was increased by a factor of about two compared to control Ringer. The voltage dependence of tau m.e.p.c. was the same in both solutions. 3. Addition of increasing amounts of CaCl2 to 2 mM-Sr2+--Ringer produced a progressive shortening of tau m.e.p.c., with no change in voltage dependence. 4. Estimates of single channel properties from noise analysis showed that the elementary conductance appeared to be slightly increased in 2 mM-Sr2+--Ringer, whilst the mean channel life-time was prolonged by a factor of about two. These changes in single channel properties are sufficient to account for the observed changes in m.e.p.c.s. 5. Following inhibition of cholinesterase activity by neostigmine, similar effects on m.e.p.c.s and single channel properties were still observed on changing to 2 mM-Sr2+--Ringer. The shapes of m.e.p.c.s in Sr2+ + neostigmine Ringer were often altered, and showed flat 'plateaus'. 6. The observed effects of Sr2+--Ringer on channel life-time cannot be explained on the basis of changes in surface charge density on the membrane, and suggest that divalent cations have an additional, and more direct, influence on receptor channel properties.

Poly(A)+ messenger RNA (mRNA) extracted from rat brains or from cat muscles was injected into Xenopus laevis oocytes. This led to the incorporation of voltage-operated Na+ and K+ channels into the oocyte membrane. These channels are not normally present in the oocyte and presumably result from the synthesis and processing of proteins coded by the injected mRNA. Tetrodotoxin blocked the Na+ channels induced by mRNA derived from either innervated or denervated muscle.

Depolarization of oocytes of Xenopus laevis usually elicits mainly passive currents, and a calcium-dependent chloride current. However, oocytes obtained from some donors show, in addition, a transient inward current on depolarization to potentials beyond ca. -40 mV. This current is abolished by tetrodotoxin at submicromolar concentrations, and is prolonged by veratrine; thus, it probably arises through sodium channels of a type similar to those found in nerve and muscle cells. However, the kinetics of the sodium currents varied between oocytes from different donors; this result suggests that genes encoding different sodium channels may be expressed in oocytes from different donors. The presence of these native channels may complicate experiments to study the expression of exogenous sodium channels encoded by foreign messenger RNAs injected into the oocyte.

Injection of inositol trisphosphate (IP3) into oocytes of Xenopus laevis induces the appearance of a transient inward (Tin) current on hyperpolarization of the membrane. This current is carried largely by chloride ions, but is shown to depend on extracellular calcium, because it is abolished by removal of calcium in the bathing fluid or by addition of manganese. Recordings with aequorin as an intracellular calcium indicator show that a calcium influx is activated by hyperpolarization after intracellular injection of IP3 as well as after activation of neurotransmitter receptors thought to mediate a rise in IP3. Furthermore, by substituting barium for calcium in the bathing solution, inward barium currents can be recorded during hyperpolarization. We conclude that intracellular IP3 modulates the activity of a class of calcium channels, so as to allow an influx of calcium on hyperpolarization. In normal Ringer solution this then leads to the generation of a chloride current, because of the large numbers of calcium-dependent chloride channels in the oocyte membrane.

Injection of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) into voltage-clamped oocytes of Xenopus laevis elicited an oscillatory chloride membrane current. This response did not depend upon extracellular calcium, because it could be produced in calcium-free solution and after addition of cobalt to block calcium channels in the surface membrane. However, it was abolished after intracellular loading with the calcium chelating agent EGTA, indicating a dependence upon intracellular calcium. The mean dose of Ins(1,3,4,5)P4 required to elicit a threshold current was 4 x 10(-14) mol. In comparison, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) gave a similar oscillatory current with doses of about one twentieth as big. Hyperpolarization of the oocyte membrane during activation by Ins(1,3,4,5)P4 elicited a transient inward current, as a result of the opening of calcium-dependent chloride channels subsequent to the entry of external calcium. In some oocytes the injection of Ins(1,3,4,5)P4 was itself sufficient to allow the generation of the transient inward current, whereas in others a prior injection of Ins(1,4,5)P3 was required. We conclude that Ins(1,3,4,5)P4 causes the release of intracellular calcium from stores in the oocyte, albeit with less potency than Ins(1,4,5)P3. In addition, Ins(1,3,4,5)P4 activates voltage-sensitive calcium channels in the surface membrane, via a process that may require 'priming' by Ins(1,4,5)P3.

1. Glutamate-activated single channels have been examined with conventional and internally perfused patch-clamp electrodes applied to the extrajunctional membrane of locust muscle fibres which were usually treated with concanavalin A to reduce desensitization. Channels opened by glutamate and other agonists have been compared.2. Recording patches were selected where there appeared to be only one active channel under the pipette. The conductance for single glutamate-activated channels was 150 pS and was not markedly dependent on clamp potential. The lifetimes of the channels were usually exponentially distributed with a mean of tau(glutamate) = 2.3 +/- 0.12 msec, T = 23 degrees C, V(m) = -60 mV.3. Channels opened by fluoroglutamate had a mean lifetime of tau(fluoroglutamate) = 1.4 +/- 0.1 msec; channels opened by quisqualate had a mean lifetime of tau(quisqualate) = 6.4 +/- 1.0 msec. The conductances of channels opened by fluoroglutamate, quisqualate and glutamate were not significantly different.4. The behaviour of individual receptor-channel complexes has been examined at various concentrations of glutamate. Drug solutions were applied through an internal perfusion pipette which allowed exchange of the solution in the patch-electrode tip within 10 sec. The distribution of channel closed times could be fitted with a single exponential. Channel lifetime was not markedly dependent on glutamate concentration (30-600 mum) whereas the channel closed time decreased with increasing glutamate concentration.5. The reciprocal of channel closed time vs. glutamate concentration had a slope value of 1.85 on logarithmic co-ordinates. The approximately second power dependence of net forward reaction rate on glutamate concentration suggests that at least two glutamate molecules activate a single receptor-channel complex.6. The apparent dissociation constant for the glutamate-receptor complex is large, being about 300-500 muM. If the receptors have an equally low affinity for neurally released transmitter, then only a small amount of the transmitter packet is expected to bind to receptors. Quisqualate and glutamate have similar receptor affinities whereas receptor affinity for fluoroglutamate is smaller.