Solution structure of peptide toxins that block mechanosensitive ion channels

Mechanosensitive channels (MSCs) play key roles in sensory processing and have been implicated as primary transducers for a variety of cellular responses ranging from osmosensing to gene expression. This paper presents the first structures of any kind known to interact specifically with MSCs. GsMTx-4 and GsMtx-2 are inhibitor cysteine knot peptides isolated from venom of the tarantula, Grammostola spatulata (Suchyna, T. M., Johnson, J. H., Hamer, K., Leykam, J. F., Gage, D. A., Clemo, H. F., Baumgarten, C. M., and Sachs, F. (2000) J. Gen. Physiol. 115, 583-598). Inhibition of cationic MSCs by the higher affinity GsMtx-4 (K(D) approximately 500 nm) reduced cell size in swollen and hypertrophic heart cells, swelling-activated currents in astrocytes, and stretch-induced arrhythmias in the heart. Despite the relatively low affinity, no cross-reactivity has been found with other channels. Using two-dimensional NMR spectroscopy, we determined the solution structure of GsMTx-4 and a lower affinity (GsMTx-2; K(D) approximately 6 microm) peptide from the same venom. The dominant feature of the two structures is a hydrophobic patch, utilizing most of the aromatic residues and surrounded with charged residues. The spatial arrangement of charged residues that are unique to GsMTx-4 and GsMTx-2 may underlie the selectivity of these peptides.     

Charybdotoxin selectively blocks small Ca-activated K channels in Aplysia neurons

The action of charybdotoxin (ChTX), a peptide component isolated from the venom of the scorpion Leiurus quinquestriatus, was investigated on membrane currents of identified neurons from the marine mollusk, Aplysia californica. Macroscopic current recordings showed that the external application of ChTX blocks the Ca-activated K current in a dose- and voltage-dependent manner. The apparent dissociation constant is 30 nM at V = -30 mV and increases e-fold for a +50- to +70-mV change in membrane potential, which indicates that the toxin molecule is sensitive to approximately 35% of the transmembrane electric field. The toxin is bound to the receptor with a 1:1 stoichiometry and its effect is reversible after washout. The toxin also suppresses the membrane leakage conductance and a resting K conductance activated by internal Ca ions. The toxin has no significant effect on the inward Na or Ca currents, the transient K current, or the delayed rectifier K current. Records from Ca-activated K channels revealed a single channel conductance of 35 +/- 5 pS at V = 0 mV in asymmetrical K solution. The channel open probability increased with the internal Ca concentration and with membrane voltage. The K channels were blocked by submillimolar concentrations of tetraethylammonium ions and by nanomolar concentrations of ChTX, but were not blocked by 4-aminopyridine if applied externally on outside-out patches. From the effects of ChTX on K current and on bursting pacemaker activity, it is concluded that the termination of bursts is in part controlled by a Ca-activated K conductance.     

Stretch induced endothelin-1 secretion by adult rat astrocytes involves calcium influx via stretch-activated ion channels (SACs)

The expression of endothelins (ETs) and ET-receptors is often upregulated in brain pathology. ET-1, a potent vasoconstrictor, also inhibits the expression of astrocyte glutamate transporters and is mitogenic for astrocytes, glioma cells, neurons, and brain capillary endothelia. We have previously shown that mechanical stress stimulates ET-1 production by adult rat astrocytes. We now show in adult astrocytes that ET-1 production is driven by calcium influx through stretch-activated ion channels (SACs) and the ET-1 production correlates with cell proliferation. Mechanical stimulation using biaxial stretch (<20%) of a rubber substrate increased ET-1 secretion, and 4 μM GsMTx-4 (a specific inhibitor of SACs) inhibited secretion by 30%. GsMTx-4 did not alter basal ET-1 levels in the absence of stretch. Decreasing the calcium influx by lowering extracellular calcium also inhibited stretch-induced ET-1 secretion without effecting ET-1 secretion in unstretched controls. Furthermore, inhibiting SACs with the less specific inhibitor streptomycin also inhibited stretch-induced ET-1 secretion. The data can be explained with a simple model in which ET-1 secretion depends on an internal Ca²⁺ threshold. This coupling of mechanical stress to the astrocyte endothelin system through SACs has treatment implications, since all pathology deforms the surrounding parenchyma.     

The mechanosensitive ion channel Piezo1 is inhibited by the peptide GsMTx4

Cells can respond to mechanical stress by gating mechanosensitive ion channels (MSCs). The cloning of Piezo1, a eukaryotic cation selective MSC, defines a new system for studying mechanical transduction at the cellular level. Because Piezo1 has electrophysiological properties similar to those of endogenous cationic MSCs that are selectively inhibited by the peptide GsMTx4, we tested whether the peptide targets Piezo1 activity. Extracellular GsMTx4 at micromolar concentrations reversibly inhibited ～80% of the mechanically induced current of outside-out patches from transfected HEK293 cells. The inhibition was voltage insensitive, and as seen with endogenous MSCs, the mirror image d enantiomer inhibited like the l. The rate constants for binding and unbinding based on Piezo1 current kinetics provided association and dissociation rates of 7.0 × 10(5) M(-1) s(-1) and 0.11 s(-1), respectively, and a K(D) of ～155 nM, similar to values previously reported for endogenous MSCs. Consistent with predicted gating modifier behavior, GsMTx4 produced an ～30 mmHg rightward shift in the pressure-gating curve and was active on closed channels. In contrast, streptomycin, a nonspecific inhibitor of cationic MSCs, showed the use-dependent inhibition characteristic of open channel block. The peptide did not block currents of the mechanical channel TREK-1 on outside-out patches. Whole-cell Piezo1 currents were also reversibly inhibited by GsMTx4, and although the off rate was nearly identical to that of outside-out patches, differences were observed for the on rate. The ability of GsMTx4 to target the mechanosensitivity of Piezo1 supports the use of this channel in high-throughput screens for pharmacological agents and diagnostic assays.     

Mechanically Activated Currents in Chick Heart Cells

As predicted from stretch-induced changes of rate and rhythm in the heart, acutely isolated embryonic chick heart cells exhibit whole-cell mechanosensitive currents. These currents were evoked by pressing on cells with a fire polished micropipette and measured through a perforated patch using a second pipette. The currents were carried by Na⁺ and K⁺ but not Cl⁻, and were independent of external Ca²⁺. The currents had linear I/V curves reversing at −16 mV and were completely blocked by Gd³⁺≥ 30 μm and Grammostola spatulata venom at a dilution of 1:1000. Approximately 20% of cells showed time dependent inactivation. In contrast to direct mechanical stimulation, hypotonic volume stress produced an increase in conductance for anions rather than cations—the two stimuli are not equivalent. The cells had two types of stretch-activated ion channels (SACs): a 21 pS nonspecific cation-selective reversing at −2 mV and a 90 pS K⁺ selective reversing at −70 mV in normal saline. The activity of SACs was strongly correlated with the presence of whole-cell currents. Both the whole-cell currents and SACs were blocked by Gd³⁺ and by Grammostola spatulata spider venom. Mechanical stimulation of spontaneously active cells increased the beating rate and this effect was blocked by Gd³⁺. We conclude that physiologically active mechanosensitive currents arise from stretch activated ion channels. Received: 8 April 1996/Revised: 8 August 1996     

Regulation of stretch-activated ANP secretion by chloride channels

This study was aimed to define roles of stretch-activated ion channels (SACs), especially Cl(-) channels, in regulation of atrial natriuretic peptide (ANP) secretion using isolated perfused beating atria. The volume load was achieved by elevating height of outflow catheter connected to isolated rat atria and the pressure load was achieved by decreasing diameter of outflow catheter. Both methods increased atrial contractility similarly although volume load was different (736microl for volume load vs. 129microl for pressure load). Atrial stretch by volume load markedly increased ECF translocation and ANP secretion but the pressure load slightly increased. The ANP secretion was positively correlated to workload generated by volume or pressure load. Treatment of atria with gadolinium, a blocker for SACs, attenuated the ECF translocation and the ANP secretion induced by volume load. A blocker for Ca2+-activated Cl(-) channel, niflumic acid (NFA), accentuated the ANP secretion induced by volume load whereas a blocker for swelling-activated Cl(-) channel, diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), attenuated the ANP secretion. The ANP secretion of hypertrophied atria by volume load was markedly reduced and the augmented effect of NFA on volume load-induced ANP secretion was not observed. These results indicate that Cl(-) channels may differently regulate stretch-activated ANP secretion.     

Spider toxins selectively block calcium currents in Drosophila

Toxins from spider venom, originally purified for their ability to block synaptic transmission in Drosophila, are potent and specific blockers of Ca2+ currents measured in cultured embryonic Drosophila neurons using the whole-cell, patch-clamp technique. Differential actions of toxins from two species of spiders indicate that different types of Drosophila neuronal Ca2+ currents can be pharmacologically distinguished. Hololena toxin preferentially blocks a non-inactivating component of the current, whereas Plectreurys toxin blocks both inactivating and non-inactivating components. These results suggest that block of a non-inactivating Ca2+ current is sufficient to block neurotransmitter release at Drosophila neuromuscular junction.     

Voltage gated ionic channels in rat cultured astrocytes, reactive astrocytes and an astrocyte-oligodendrocyte progenitor cell

Astrocytes (both type 1 and type 2), cultured from the central nervous system of newborn or 7 day old rats show voltage gated sodium and potassium channels that are activated when the membrane is depolarized to greater than -40 mV. The sodium channels in these cells have an h-infinity curve similar to that of nodal membranes but the activation (peak current-voltage) curves are shifted along the voltage axis by about +30 mV. These sodium currents are blocked only by high concentrations of tetrodotoxin. The voltage activated potassium currents in both types of astrocyte show at least two components; an inactivating component that is suppressed at holding potentials of greater than -40 mV and a persistent, non-inactivating current. Several types of single channel currents were observed in outside-out membrane patches from type 2 astrocytes. One type of potassium channel showed inactivation on depolarization and may contribute to the whole-cell inactivating current. In contrast, oligodendrocytes showed no obvious voltage gated membrane channels. The properties of the type 2 astrocyte-oligodendrocyte progenitor cell were investigated in two ways: 1) by examination of cells just beginning to differentiate along the "electrically silent" oligodendrocyte pathway or 2) by recording from progenitor cells cultured for 24 hours in the presence of cycloheximide to block the appearance of new membrane channels. In both cases, voltage gated inward (sodium) and outward (potassium) currents were noted. The outward current response showed both an inactivating and a non-inactivating component. Similar voltage activated inward and outward membrane currents were noted in reactive astrocytes freshly isolated (3-6 hours) from lesioned areas of adult rat brains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.     

Isolation and pharmacological characterisation of delta-atracotoxin-Hv1b, a vertebrate-selective sodium channel toxin

delta-Atracotoxins (delta-ACTXs) are peptide toxins isolated from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion alpha-toxins. We have isolated and determined the amino acid sequence of a novel delta-ACTX, designated delta-ACTX-Hv1b, from the venom of the funnel-web spider Hadronyche versuta. This 42 residue toxin shows 67% sequence identity with delta-ACTX-Hv1a previously isolated from the same spider. Under whole-cell voltage-clamp conditions, the toxin had no effect on tetrodotoxin (TTX)-resistant sodium currents in rat dorsal root ganglion neurones but exerted a concentration-dependent reduction in peak TTX-sensitive sodium current amplitude accompanied by a slowing of sodium current inactivation similar to other delta-ACTXs. However, delta-ACTX-Hv1b is approximately 15-30-fold less potent than other delta-ACTXs and is remarkable for its complete lack of insecticidal activity. Thus, the sequence differences between delta-ACTX-Hv1a and -Hv1b provide key insights into the residues that are critical for targeting of these toxins to vertebrate and invertebrate sodium channels.     

BmP09, a "long chain" scorpion peptide blocker of BK channels

A novel "long chain" toxin BmP09 has been purified and characterized from the venom of the Chinese scorpion Buthus martensi Karsch. The toxin BmP09 is composed of 66 amino acid residues, including eight cysteines, with a mass of 7721.0 Da. Compared with the B. martensi Karsch AS-1 as a Na(+) channel blocker (7704.8 Da), the BmP09 has an exclusive difference in sequence by an oxidative modification at the C terminus. The sulfoxide Met-66 at the C terminus brought the peptide a dramatic switch from a Na(+) channel blocker toaK(+) channel blocker. Upon probing the targets of the toxin BmP09 on the isolated mouse adrenal medulla chromaffin cells, where a variety of ion channels coexists, we found that the toxin BmP09 specifically blocked large conductance Ca(2+)- and voltage-dependent K(+) channels (BK) but not Na(+) channels at a range of 100 nm concentration. This was further confirmed by blocking directly the BK channels encoded with mSlo1 alpha-subunits in Xenopus oocytes. The half-maximum concentration EC(50) of BmP09 was 27 nm, and the Hill coefficient was 1.8. In outside-out patches, the 100 nm BmP09 reduced approximately 70% currents of BK channels without affecting the single-channel conductance. In comparison with the "short chain" scorpion peptide toxins such as Charybdotoxin, the toxin BmP09 behaves much better in specificity and reversibility, and thus it will be a more efficient tool for studying BK channels. A three-dimensional simulation between a BmP09 toxin and an mSlo channel shows that the Lys-41 in BmP09 lies at the center of the interface and plugs into the entrance of the channel pore. The stable binding between the toxin BmP09 and the BK channel is favored by aromatic pi -pi interactions around the center.     

Gamma-dendrotoxin blocks large conductance Ca2+-activated K+ channels in neuroblastoma cells

In N1E 115 neuroblastoma cells, gamma-dendrotoxin (DTX, 200 nM) blocked the outward K(+) current by 31.1 +/- 3.5% (n = 4) with approximately 500 nM Ca(2+) in the pipet solution, but had no effect on the outward K(+) current when internal Ca(2+) was reduced. Using a ramp protocol, iberiotoxin (IbTX, 100 nM) inhibited a component of the whole cell current, but in the presence of 200 nM gamma-DTX, no further inhibition by IbTX was observed. Two types of single channels were seen using outside-out patches when the pipette free Ca(2+) concentration was approximately 500 nM; a 63 pS and a 187 pS channel. The 63 pS channel was TEA-, IbTX- and gamma-DTX-insensitive, while the 187 pS channel was blocked by 1 mM TEA, 100 nM IbTX or 200 nM gamma-DTX. Both channels were activated by external application of ionomycin, when the pipet calcium concentration was reduced. gamma-DTX (200 nM) reduced the probability of openings of the 187 pS channel, with an IC(50) of 8.5 nM. In GH(3) cells gamma-DTX (200 nM) also blocked an IbTX-sensitive component of whole-cell K(+) currents. These results suggest that gamma-DTX blocks a large conductance Ca(2+) activated K(+) current in N1E 115 cells. This is the first indication that any of the dendrotoxins, which have classically been known to block voltage-gated (Kv) channels, can also block Ca(2+) activated K(+) channels.     

Action of tetraethylammonium on calcium-activated potassium channels in pig pancreatic acinar cells studied by patch-clamp single-channel and whole-cell current recording

Summary The effects of tetraethylammonium ions on currents through high-conductance voltage- and Ca²⁺-activated K⁺ channels have been studied with the help of patch-clamp single-channel and whole-cell current recording on pig pancreatic acinar cells. In excised outside-out membrane patches TEA (1 to 2 mM) added to the bath solution virtually abolishes unitary current activity except at very positive membrane potentials when unitary currents corresponding to a markedly reduced conductance are observed. TEA in a lower concentration (0.2 mM) markedly reduces the open-state probability and causes some reduction of the single-channel conductance. In inside-out membrane patches bath application of TEA in concentrations up to 2 mM has no effect on single-channel currents. At a higher concentration (10 mM) slight reductions in single-channel conductance occur. In whole-cell current recording experiments TEA (1 to 2 mM) added to the bath solution completely suppresses the outward currents associated with depolarizing voltage jumps to membrane potentials of 0 mV and blocks the major part (70 to 90%) of the outward currents even at very positive membrane potentials (30 to 40 mV). In contrast TEA (2 mM) added to the cell interior (pipette solution) has no effect on the outward K⁺ current. Our results demonstrate that TEA in low concentrations (1 to 2 mM) acts specifically on the outside of the plasma membrane to block current through the high-conductance Ca²⁺- and voltage-activated K⁺ channels     

Isolation and pharmacological characterisation of δ-atracotoxin-Hv1b, a vertebrate-selective sodium channel toxin

δ-Atracotoxins (δ-ACTXs) are peptide toxins isolated from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion α-toxins. We have isolated and determined the amino acid sequence of a novel δ-ACTX, designated δ-ACTX-Hv1b, from the venom of the funnel-web spider Hadronyche versuta. This 42 residue toxin shows 67% sequence identity with δ-ACTX-Hv1a previously isolated from the same spider. Under whole-cell voltage-clamp conditions, the toxin had no effect on tetrodotoxin (TTX)-resistant sodium currents in rat dorsal root ganglion neurones but exerted a concentration-dependent reduction in peak TTX-sensitive sodium current amplitude accompanied by a slowing of sodium current inactivation similar to other δ-ACTXs. However, δ-ACTX-Hv1b is approximately 15–30-fold less potent than other δ-ACTXs and is remarkable for its complete lack of insecticidal activity. Thus, the sequence differences between δ-ACTX-Hv1a and -Hv1b provide key insights into the residues that are critical for targeting of these toxins to vertebrate and invertebrate sodium channels.     

Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units.     

Pandinus imperator scorpion venom blocks voltage-gated potassium channels in GH3 cells

We examined the effects of Pandinus imperator scorpion venom on voltage-gated potassium channels in cultured clonal rat anterior pituitary cells (GH3 cells) using the gigohm-seal voltage-clamp method in the whole-cell configuration. We found that Pandinus venom blocks the voltage-gated potassium channels of GH3 cells in a voltage-dependent and dose-dependent manner. Crude venom in concentrations of 50-500 micrograms/ml produced 50-70% block of potassium currents measured at -20 mV, compared with 25-60% block measured at +50 mV. The venom both decreased the peak potassium current and shifted the voltage dependence of potassium current activation to more positive potentials. Pandinus venom affected potassium channel kinetics by slowing channel opening, speeding deactivation slightly, and increasing inactivation rates. Potassium currents in cells exposed to Pandinus venom did not recover control amplitudes or kinetics even after 20-40 min of washing with venom-free solution. The concentration dependence of crude venom block indicates that the toxins it contains are effective in the nanomolar range of concentrations. The effects of Pandinus venom were mimicked by zinc at concentrations less than or equal to 0.2 mM. Block of potassium current by zinc was voltage dependent and resembled Pandinus venom block, except that block by zinc was rapidly reversible. Since zinc is found in crude Pandinus venom, it could be important in the interaction of the venom with the potassium channel. We conclude that Pandinus venom contains toxins that bind tightly to voltage-dependent potassium channels in GH3 cells. Because of its high affinity for voltage-gated potassium channels and its irreversibility, Pandinus venom may be useful in the isolation, mapping, and characterization of voltage-gated potassium channels.     

High-threshold mechanosensitive ion channels blocked by a novel conopeptide mediate pressure-evoked pain

Little is known about the molecular basis of somatosensory mechanotransduction in mammals. We screened a library of peptide toxins for effects on mechanically activated currents in cultured dorsal root ganglion neurons. One conopeptide analogue, termed NMB-1 for noxious mechanosensation blocker 1, selectively inhibits (IC(50) 1 microM) sustained mechanically activated currents in a subset of sensory neurons. Biotinylated NMB-1 retains activity and binds selectively to peripherin-positive nociceptive sensory neurons. The selectivity of NMB-1 was confirmed by the fact that it has no inhibitory effects on voltage-gated sodium and calcium channels, or ligand-gated channels such as acid-sensing ion channels or TRPA1 channels. Conversely, the tarantula toxin, GsMTx-4, which inhibits stretch-activated ion channels, had no effects on mechanically activated currents in sensory neurons. In behavioral assays, NMB-1 inhibits responses only to high intensity, painful mechanical stimulation and has no effects on low intensity mechanical stimulation or thermosensation. Unexpectedly, NMB-1 was found to also be an inhibitor of rapid FM1-43 loading (a measure of mechanotransduction) in cochlear hair cells. These data demonstrate that pharmacologically distinct channels respond to distinct types of mechanical stimuli and suggest that mechanically activated sustained currents underlie noxious mechanosensation. NMB-1 thus provides a novel diagnostic tool for the molecular definition of channels involved in hearing and pressure-evoked pain.     

Voltage-Induced Activation of Mechanosensitive Cation Channels of Leech Neurons

The voltage dependence of stretch-activated cation channels in leech central neurons was studied in cell-free configurations of the patch-clamp technique. We established that stretch-activated channels excised from identified cell bodies of desheathed ganglia, as well as from neurons in culture, were slowly and reversibly activated by depolarizing membrane potentials. Negative pressure stimuli, applied to the patch pipette during a slow periodical modulation of membrane potential, enhanced channel activity, whereas positive pressures depressed it. Voltage-induced channel activation was observed, with soft glass pipettes, both in inside-out and outside-out membrane patches, at negative and positive reference potentials, respectively. The results presented in this study demonstrate that membrane depolarization induces slow activation of stretch-activated channels of leech central neurons. This phenomenon is similar to that found in Xenopus oocytes, however, some peculiar features of the voltage dependence in leech stretch-activated channels indicate that specific membrane-glass interactions might not necessarily be involved. Moreover, following depolarization, stretch-activated channels in membrane patches from neurons in culture exhibited significantly shorter delay to activation (sec) than their counterparts from neurons of freshly isolated ganglia (hundreds of sec).     

Volume-regulated anion and organic osmolyte channels in mouse zygotes

Whole-cell currents in mouse zygotes were measured using the patch-clamp technique in whole-cell mode. Upon exposure to hypotonic medium, patch-clamped zygotes increased in volume and developed a large swelling-activated current. The swelling-activated current was blocked by Cl- channel blockers, and the magnitude of the current and reversal potential were dependent on the Cl- gradient. Thus, the swelling-activated current had the properties of a current mediated by anion channels. However, in addition to being permeable to Cl- and I- (with I- having the greater permeability), there was also a significant swelling-activated conductance to aspartate and taurine, indicating that the swelling-activated channels in zygotes conduct not only inorganic anions but organic osmolytes as well. This swelling-activated anion and organic osmolyte pathway likely underlies the ability of zygotes to recover from an increase in volume, and it may function to regulate intracellular amino acid concentrations.