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O P Hamill

Publications and source records attributed to O P Hamill.

At least 19 recordsLinked to original sources
PubMed2001

Brefeldin A block of integrin-dependent mechanosensitive ATP release from Xenopus oocytes reveals a novel mechanism of mechanotransduction.

Many animal cells release ATP into the extracellular medium, and often this release is mechanosensitive. However, the mechanisms underlying this release are not well understood. Using the luciferin-luciferase bioluminescent assay we demonstrate that a Xenopus oocyte releases ATP at a basal rate approximately 0.01 fmol/s, and gentle mechanical stimulation can increase this to 50 fmol/s. Brefeldin A, nocodazole, and progesterone-induced- maturation block basal and mechanosensitive ATP release. These treatments share the common feature of disrupting the Golgi complex and vesicle trafficking to the cell surface and thereby block protein secretion and membrane protein insertion. We propose that ATP release occurs when protein transport vesicles enriched in ATP fuse with the plasma membrane. Collagenase, integrin-binding peptides, and cytochalasin D also block ATP release, indicating that extracellular, membrane and cytoskeletal elements are involved in the release process. Elevation of intracellular Ca(2+) does not evoke ATP release but potentiates mechanosensitive ATP release. Our study indicates a novel mechanism of mechanotransduction that would allow cells to regulate membrane trafficking and protein transport/secretion in response to mechanical loading.

Adenosine Triphosphate↗
PubMed2001

Molecular basis of mechanotransduction in living cells.

The simplest cell-like structure, the lipid bilayer vesicle, can respond to mechanical deformation by elastic membrane dilation/thinning and curvature changes. When a protein is inserted in the lipid bilayer, an energetic cost may arise because of hydrophobic mismatch between the protein and bilayer. Localized changes in bilayer thickness and curvature may compensate for this mismatch. The peptides alamethicin and gramicidin and the bacterial membrane protein MscL form mechanically gated (MG) channels when inserted in lipid bilayers. Their mechanosensitivity may arise because channel opening is associated with a change in the protein's membrane-occupied area, its hydrophobic mismatch with the bilayer, excluded water volume, or a combination of these effects. As a consequence, bilayer dilation/thinning or changes in local membrane curvature may shift the equilibrium between channel conformations. Recent evidence indicates that MG channels in specific animal cell types (e.g., Xenopus oocytes) are also gated directly by bilayer tension. However, animal cells lack the rigid cell wall that protects bacteria and plants cells from excessive expansion of their bilayer. Instead, a cortical cytoskeleton (CSK) provides a structural framework that allows the animal cell to maintain a stable excess membrane area (i.e., for its volume occupied by a sphere) in the form of membrane folds, ruffles, and microvilli. This excess membrane provides an immediate membrane reserve that may protect the bilayer from sudden changes in bilayer tension. Contractile elements within the CSK may locally slacken or tighten bilayer tension to regulate mechanosensitivity, whereas membrane blebbing and tight seal patch formation, by using up membrane reserves, may increase membrane mechanosensitivity. In specific cases, extracellular and/or CSK proteins (i.e., tethers) may transmit mechanical forces to the process (e.g., hair cell MG channels, MS intracellular Ca(2+) release, and transmitter release) without increasing tension in the lipid bilayer.

Amino Acid Sequence↗
PubMed2000

On the discrepancy between whole-cell and membrane patch mechanosensitivity in Xenopus oocytes.

1. Mechanical stimulation of voltage-clamped Xenopus oocytes by inflation, aspiration, or local indentation failed to activate an increase in membrane conductance up to the point of causing visible oocyte damage. 2. The absence of mechanosensitivity is not due to the vitelline membrane, rapid MG channel adaptation or tension-sensitive recruitment of new membrane. 3. Membrane capacitance measurements indicate that the oocyte surface area is at least 5 times larger than that predicted assuming a smooth sphere. We propose that this excess membrane area provides an immediate reserve that can 'buffer' membrane tension changes and thus prevent MG channel activation. 4. High-resolution images of tightly sealed patches and patch capacitance measurements indicate a smooth membrane that is pulled flat and perpendicular across the inside of the pipette. Brief steps of pressure or suction cause rapid and reversible membrane flexing and MG channel activation. 5. We propose that changes in membrane geometry induced during cell growth and differentiation or as a consequence of specific physiological and pathological conditions may alter mechanosensitivity of a cell independently of the intrinsic properties of channel proteins.

Adaptation, Physiological↗
PubMed2000

Mechanically gated channel activity in cytoskeleton-deficient plasma membrane blebs and vesicles from Xenopus oocytes.

1. A novel technique involving hypertonic stress causes membrane 'blebbing' of the Xenopus oocyte and the shedding of plasma membrane vesicles (PMVs). 2. Confocal fluorescence microscopy, immunocytochemistry and electron microscopy indicate that blebs and PMVs lack cortical cytoskeleton and are deficient in cytoskeleton proteins and devoid of microvilli. 3. Patch recordings from PMVs consistently reveal mechanically gated (MG) channel activity. The MG channels display the same single-channel conductance as control recordings but differ in terms of reduced mechanosensitivity and adaptation to sustained stimulation. 4. Whole PMV recordings show rapid and reversible activation of mechanosensitive currents in response to pressure pulses. The maximal currents activated in PMVs are consistent with MG channel activity recorded in patches. 5. The discrepancy between MG channel activity recorded in whole PMVs and oocytes most probably reflects their different membrane geometry and ability to develop activating bilayer tensions. 6. We propose that membrane blebbing, which is known to occur under specific physiological and pathological conditions (e.g. mitosis and apoptosis), may increase mechanosensitivity independently of the intrinsic properties of membrane proteins.

Animals↗
PubMed2000

Calcium-, voltage- and osmotic stress-sensitive currents in Xenopus oocytes and their relationship to single mechanically gated channels.

1. Patch recordings from Xenopus oocytes indicated that mechanically gated (MG) channels are expressed at a uniform surface density ( approximately 1 channel microm-2) with an estimated > 3 x 106 MG channels per oocyte that could generate microamps of current at +/-50 mV. 2. Removal of external Ca2+ induced a membrane conductance that differed from MG channels in ion selectivity, pharmacology and sensitivity to connexion-38. 3. Depolarization to +50 mV activated a Na+-selective, a Cl--selective and a non-selective conductance. Hyperpolarization to -150 mV activated a non-selective conductance. None of these conductances appeared to be mediated by MG channels. 4. Hypotonicity (25 %) failed to evoke any change in membrane conductance in the majority of defolliculated oocytes. Hypertonicity (200 %) evoked a large non-selective (PK /PCl approximately 1) membrane conductance that was not blocked by 100 microM Gd3+. 5. Although the above stimuli could activate a variety of whole-oocyte conductances, including three novel conductances, they did not involve MG channel activation. Possible mechanisms underlying the discrepancy between observed conductances and those anticipated from patch-clamp studies are discussed.

Animals↗
PubMed1998

Effects of mechano-gated cation channel blockers on Xenopus oocyte growth and development.

The putative role(s) of a mechanically gated (MG) cation channel in Xenopus oocyte growth, maturation, fertilization and embryogenesis has been examined. Using a pharmacological approach, we have tested the effects of the MG channel blockers, gadolinium, gentamicin and amiloride on the above developmental events. Our results indicate that oocyte maturation, fertilization and early embryogenesis (up to the free-swimming stage 45) can proceed normally in the presence of concentrations of agents that either completely abolish (i.e., > or = 10 microM Gd3+) or partially block (i.e., 1 mM gentamicin) single MG channel activity as measured by patch-clamp recording. However, we also find that higher concentrations of Gd3+ (> or = 50 microM) can lead to an increased percentage (> 20%) of axis-perturbed embryos compared with control (< 1%) and that amiloride (0.5 mM) reduces the success of fertilization (from 100% to < 50%) and increases mortality (by approximately 75%) in developing embryos. Furthermore, we find that all three agents inhibit oocyte growth in vitro. However, their order of effectiveness (amiloride > gentamicin > Gd3+) is opposite to their order for blocking MG channels (Gd3+ >> gentamicin > amiloride). These discrepancies indicated that the drugs effects occur by mechanisms other than, or in addition to, MG channel block. Our results provide no compelling evidence for the idea that MG channel activity is critical for development in Xenopus. This could mean that there are other mechanisms in the oocyte that can compensate when MG channel activity is blocked or that the protein that forms the channel can undergo additional interactions that result in a function insensitive to MG channel blockers.

Amiloride↗
PubMed1998

The ion selectivity of a membrane conductance inactivated by extracellular calcium in Xenopus oocytes.

1. The ion selectivity of a membrane ion conductance that is inactivated by extracellular calcium (Ca2+o) in Xenopus oocytes has been studied using the voltage-clamp technique. 2. The reversal potential of the Ca2+o-sensitive current (Ic) was measured using voltage ramps (-80 to +40 mV) as a function of the external concentration (12-240 mM) of NaCl or KCl. The direction and amplitude of the shifts in reversal potentials are consistent with permeability ratios of 1:0.99:0.24 for K+:Na+:Cl-. 3. Current-voltage (I-V ) relations of Ic, determined during either voltage ramps of 0.5 s duration or at steady state, displayed pronounced rectification at both hyperpolarized and depolarized potentials. However, instantaneous I-V relations showed less rectification and could be fitted by the constant field equation assuming the above K+:Na+:Cl- permeability ratios. 4. Ion substitution experiments indicated that relatively large organic monovalent cations and anions are permeant through Ic channels with the permeability ratios K+:NMDG+:TEA+:TPA+:TBA+:Gluc- = 1:0.45:0. 35:0.2:0.2:0.2. 5. External amiloride (200 microM), gentamicin (220 microM), flufenamic acid (40 microM), niflumic acid (100 microM), Gd3+ (0.3 microM) or Ca2+ (200 microM) caused reversible block of Ic without changing its reversal potential. 6. Preinjection of oocytes with antisense oligonucleotide against connexin 38, the Xenopus hemi-gap-junctional protein, inhibited Ic by 80 % without affecting its ion selectivity, thus confirming and extending the recent suggestion of Ebihara that Ic represents current carried through hemi-gap-junctional channels. 7. In vitro and in vivo maturation of oocytes resulted in a significant decrease in Ic conductance to 7 % and 2 % of control values, respectively. This developmental downregulation of Ic minimizes any toxic effect Ic activation would have when the mature egg is released into Ca2+o-free pond water. 8. The results of this study are discussed in relation to other Ca2+o-inactivated conductances seen in a wide variety of cell types and which have previously been interpreted as arising either from Ca2+o-masked channels or from changes in the ion selectivity of voltage-gated Ca2+ or K+ channels.

Amiloride↗
PubMed1998

Mg2+ block and inward rectification of mechanosensitive channels in Xenopus oocytes.

The effects of Mg2+ on single mechanosensitive (MS) channel currents recorded from Xenopus oocytes were studied using cell-attached and inside-out patch configurations. Mg2+ both permeates and blocks MS channels. Under symmetrical ionic conditions, the blocking effects of Mg2+ can be described by a Hill coefficient of 0.9 at +/-100 mV and IC50s of 0.12 mM (-100 mV) and 0.60 mM at (+100 mV). Although block by intracellular Mg2+ may contribute to inward MS channel rectification, significant current rectification is retained even under symmetrical KCl concentrations and in the complete absence of Mg2+. The observed voltage dependencies of the IC50 for Mg2+ block and the Km for K+ current saturation indicate asymmetries in the MS channel pore. In addition, the absence of K+ self block and anomalous mole fraction effects with K+/Tl+ mixtures indicate a single site pore model.

Animals↗
PubMed1997

Induced membrane hypo/hyper-mechanosensitivity: a limitation of patch-clamp recording.

Practical limitations of the patch-clamp technique when recording mechanogated membrane ion channels are considered. Mechanical overstimulation of the patch or the cell from excessive suction/pressure protocols induces morphological and functional changes. In particular, the plasma membrane becomes decoupled from the underlying cytoskeleton to form either membrane blebs (cell-attached) or ghosts (whole cell). As a consequence, a membrane ion channel may show either a decrease or an increase in its native mechanosensitivity or even acquire mechanosensitivity. The effect varies with ion channel and cell type and presumably arises because of a disruption of membrane-cytoskeleton interactions. We consider that such disruptions are a pathological consequence of excessive mechanical stress, either during or after seal formation, rather than an immutable consequence of patch-clamp recording. By careful attention to the suction/pressure protocols during sealing and throughout recording, such artifacts can be avoided.

Animals↗
PubMed1996

The pharmacology of mechanogated membrane ion channels.

In this article, the actions, mechanisms and applications of various ions and drugs that interact with MG channels have been discussed. At present, no compound has been found that displays the high specificity and affinity exhibited by tetrodotoxin or alpha-bungarotoxin that proved so useful in the functional and structural characterization of the voltage-gated Na+ channel and the acetylcholine receptor channel, respectively. Nevertheless, three different classes of compounds have been discovered since Paintal's review that clearly block MG channels. These compounds, represented by amiloride, gentamicin and gadolinium, act mainly on the SA cation channel, which appears to be shared by many nonsensory and some mechanosensory cells. Each class of compound can be distinguished by the voltage and concentration dependence of the block and most likely involves different mechanisms of blocking action. In general, the MG channel blocker pharmacology indicates a variety of "receptor sites" on MG channels. The recognition and acceptance of such receptors should provide added impetus for continued screening for more potent drugs, venoms and toxins. In the case of activators, little is understood of the mechanisms by which the various amphipathic and amphiphilic compounds stimulate MG channels, although different bilayer and protein mechanisms have been evoked. Even less is understood of the role the new class of MG K+ channel and their modulation by fatty acids plays in physiological and perhaps pathological processes. However, given that K+ channels in general tend to reduce the excitability of nerve and muscle, plausible roles include fatty acid regulation of vascular tone and control of neuronal network excitability. In both cases, more detailed understanding is required regarding the physiological stimuli that modulate these channels through their fatty acid receptors. It may turn out that recognition and/or development of cell-type specific agents that activate such MG channels will possess high therapeutic potential. In any case, the observation that MG channels can be chemically blocked and/or activated by a wide range of compounds requires revision of the long-standing conclusion of Paintal that mechanotransduction is a process that has a low susceptibility to chemical influence.

Amiloride↗
PubMed1994

Mouse muscle epsilon- and gamma-containing acetylcholine receptors expressed in Xenopus laevis oocytes do not differ in their degradation half-lives.

Mouse acetylcholine receptors (AChRs) consisting of either the embryonic form (2 alpha, 1 beta, 1 delta and 1 gamma) or the adult form (2 alpha, 1 beta, 1 delta and 1 epsilon) were expressed in Xenopus laevis oocytes. As expected, the single channel conductance was approximately 52 pS and the exponential decay time constants were 2.2 and 8.2 ms for the gamma-AChR and respectively 65 pS and 0.6 and 2.8 ms for the epsilon-AChR. No difference was seen in the degradation rate between the gamma- and epsilon-containing AChRs, both having a half-life of about 5 days.

Animals↗
PubMed1993

Pressure-clamp technique for measurement of the relaxation kinetics of mechanosensitive channels.

The pressure-clamp technique, used in conjunction with patch-clamp techniques, allows the application of precise pressure/suction waveforms to membrane patches and whole cells. Using step perturbations in pressure, it allows rapid relaxation measurements of the latency, turn-on, turn-off and adaptation kinetics of mechanosensitive membrane ion channels. The pressure-clamp technique also provides the ability to apply gentle and reproducible sealing protocols to establish tight seals and thereby minimize membrane-cytoskeleton disruption which can otherwise alter channel properties.

Animals↗
PubMed1993

Ionic effects on amiloride block of the mechanosensitive channel in Xenopus oocytes.

1. Patch clamp techniques were used to measure the ionic dependence of amiloride block of single mechanosensitive (MS) channels in frog (Xenopus laevis) oocytes. 2. The primary aim was to determine whether the difference in potency of amiloride block of MS channels in frog oocytes (IC50 = 0.5 mM) and chick auditory hair cells (IC50 = 50 microM) was due to the different ionic recording solutions. 3. Amiloride block of the oocyte MS channel does not vary significantly with complete substitution of external Na+ (i.e. 100 mM) with K+ in Ca(2+)-free recording solution (in both Na+ and K+ the IC50 = 0.5 mM). 4. A physiological concentration (1.8 mM) of external Ca2+ blocks the oocyte MS channel and reduces the potency of amiloride block (IC50 = 1.1 mM) without altering the voltage-dependence or the HIll coefficient (n = 1.8) of amiloride block. The reduction in potency can be explained by surface charge screening by Ca2+ which reduces the effective amiloride surface concentration. 5. The present results indicate that factors other than ionic recording conditions must underlie the difference in potency of amiloride block of MS channels in oocytes and auditory hair cells.

Amiloride↗