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D W McBride

Publications and source records attributed to D W McBride.

At least 19 recordsLinked to original sources
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↗
PubMed1997

A quantitative analysis of passive electrolocation behavior in electric fish.

Weakly electric fish of the families Gymnotidae and Hypopomidae (Gymnotiformes) are able to locate the electric discharges from conspecifics or from dipole electrodes, and they demonstrate this by making rapid, well-directed approaches toward these electrical sources. A video tracking system was used to follow the movements of electric fish in a large tank and an analytic method was used for computing the direction and magnitude of the electric field anywhere within the cylindrical test tank. Using a static analysis method, we describe the posture of test fish relative to the electric fields during their approaches to stationary or moving electrical stimuli. Using a dynamic analysis, we examine the movements of the fish including the sign and magnitude of velocity and bending in response to electric fields. Electric fish seek to maintain a zero error angle between their body orientation and the local electric field. They do so by bending their body in the direction of the local electric field. The response has a delay of approximately 0.5 s. Swimming in reverse inverts the direction of the bend. These fish also use 'V-turns' to redirect their swim directions when encountering rapidly-changing electric fields.

Animals↗
PubMed1996

Short-range orientation in electric fish: an experimental study of passive electrolocation.

Gymnotiform electric fish are capable of locating and approaching an electrically discharging conspecific over a range of 1-2 m in a behavior called passive electrolocation. This paper investigates the movements of two species in experiments with approaches to stationary dipoles that are either silenced or jumped to a new direction during an approach. Gymnotus carapo fail to find an electrode source in trials in which the dipole electrode is switched off in mid-track. They slow their approach, become disoriented and drift away from the target within seconds of the field being switched off. This result suggests that the fish are unable to construct a cognitive map of a dipole source from brief exposure to local electrosensory stimuli. The second set of trials shows that Brachyhypopomus diazi and Gymnotus carapo bend their body to track electric vectors which are suddenly jumped to a new direction. The latency of the bend response is 0.5 s after the jump. Bending initiates a turn that reduces to zero the error between the fish's direction and the electric field vector and helps keep the fish aligned with the local electric field vector. Together, these experiments suggest that passive electrolocation is stimulus-bound and that these fish find the electrical sources simply by tracking instantaneous local electric current vectors.

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↗
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↗
PubMed1992

Rapid adaptation of single mechanosensitive channels in Xenopus oocytes.

Mechanosensitive (MS) channels are expressed in a wide range of cell types and have been implicated in diverse functions, including osmoregulation and mechanoreception. The majority of previous studies on single MS channels have been carried out on nonsensory cells and have dealt with the steady-state properties of the channel. Here we measure the dynamic or nonstationary properties of the MS channel in Xenopus laevis oocytes. MS channels open transiently in response to a step change in suction applied to the membrane patch. This adaptive behavior occurs because of a reduction in open channel probability rather than a decrease in channel conductance. Double-step suction protocols indicate that adapted MS channels can be reactivated by application of stronger stimulation, consistent with a change in gating sensitivity rather than channel inactivation. Adaptation is highly voltage dependent, being most evident at resting or hyperpolarized potentials and absent at strongly positive potentials. Neither adaptation nor its voltage sensitivity requires the presence of extracellular Ca2+. Adaptation is fragile, dependent on patch history, and can be irreversibly abolished by moderate suction applied to the patch while MS channel activity is retained. Further suction can abolish MS channel activity without compromising the seal. We propose that the selective loss of adaptation and MS channel activity is due to different stages of membrane-cytoskeleton decoupling caused by the mechanical stresses associated with patch clamp recording.

Acclimatization↗
PubMed1992

Pressure-clamp: a method for rapid step perturbation of mechanosensitive channels.

Here we describe a pressure-clamp method for applying suction or pressure steps to membrane patches in order to study the activation, adaptation and relaxation characteristics of mechanosensitive (MS) channels. A description is given of the mechanical arrangement of the pressure clamp which involves a balance between negative (suction) and positive pressures. The electronic circuitry of the feedback control is described. We also describe the optimal time response (approximately 10 ms) of the pressure-clamp, the amplitude of pressure resolution (0.2-0.5 mmHg; 27-67 Pa) and the factors influencing these parameters. We illustrate the applications of the clamp on the Xenopus oocyte and cultured skeletal myotubes from dystrophic mouse (mdx) muscle, both of which express MS channels. Studies with pressure/suction pulses indicate that in both muscles and oocytes MS channel activity displays adaptation. The ability to study current relaxations following step changes in pressure/suction using the pressure-clamp in combination with patch-clamp techniques provides the opportunity for analysis of the time, voltage and pressure dependence of the opening and closing of MS channels.

Animals↗
PubMed1992

Structure-activity relations of amiloride and its analogues in blocking the mechanosensitive channel in Xenopus oocytes.

1. Patch clamp recording techniques have been used to compare the block caused by amiloride and some of its structural analogues of the mechanosensitive (MS) cation selective channel in frog (Xenopus laevis) oocytes. 2. Like amiloride, the amiloride analogues dimethylamiloride (DMA), benzamil and bromohexamethyleneamiloride (BrHMA) block the MS channel in a highly voltage-dependent manner. 3. All analogues tested were more potent blockers than amiloride with IC50's of 500 microM (amiloride), 370 microM (DMA), 95 microM (benzamil) and 34 microM (BrHMA). 4. Hill plots gave Hill coefficients of 2 (amiloride), 1.8 (DMA), 1 (benzamil) and 1.2 (BrHMA) indicating that the binding of two ligand molecules may be necessary for the block caused by amiloride, DMA and possibly BrHMA whereas only a single ligand molecule may be required for the block by benzamil. 5. The potential use of BrHMA as a light-activated, covalent label of the MS channel protein is discussed. 6. The amiloride analogue 'fingerprinting' of the blocking site on the MS channel indicates it is structurally different from previously described amiloride-sensitive ion transport pathways but may be related to the amiloride binding site on outer hair cells of the ear.

Amiloride↗
PubMed1991

Ca(2+)-dependent chloride conductance in Necturus taste cells.

This report describes the occurrence and localization of a Ca(2+)-dependent chloride conductance in taste cells of Necturus maculosus. Lingual epithelium from Necturus was removed with blunt dissection and mounted in a modified Ussing chamber which allowed individual taste cells to be impaled with intracellular micropipettes. Solutions in the mucosal and serosal chambers could be changed independently and the properties of apical and basolateral membranes tested separately. Action potentials in taste cells, elicited by brief depolarizing current pulses passed through the intracellular recording microelectrode, provided an accurate description of whether voltage-dependent conductances had been blocked or unmasked by the experimental conditions. We found that Ca2+ influx during the action potential triggers a prolonged depolarization due to Ca(2+)-dependent conductance changes, particularly in the presence of TEA to block repolarizing K+ currents. This afterdepolarization could last up to 7 sec and is due, in part, to a Ca(2+)-dependent Cl- conductance. Other Ca(2+)-dependent channels such as Ca(2+)-dependent K+ channels or nonselective cation channels may also contribute to the afterpotential. Calcium-dependent conductance channels were situated on apical and basolateral membranes of the taste cells. We speculate that Ca(2+)-dependent Cl- channels may play a role in discriminating chloride salts from salts of other anions and may help shape receptor cell responses elicited by taste stimuli.

Action Potentials↗
PubMed1991

Amiloride block of the mechanosensitive cation channel in Xenopus oocytes.

1. Patch clamp recording techniques have been used to investigate the block by amiloride of the mechanosensitive cation-selective channel in frog (Xenopus laevis) oocytes. 2. Cell-attached and outside-out patch recording configurations were employed to study the differences in block produced when amiloride was present at either the extracellular (external) or intracellular (internal) membrane face. 3. External amiloride causes a highly voltage-dependent 'flickery' block of single mechanosensitive channel currents in which inward mechanosensitive current recorded at negative potentials is reduced in amplitude but outward mechanosensitive current recorded at positive potentials is almost unaffected. 4. At -100 mV the apparent dissociation constant (Kd) for external amiloride block is 0.5 mM. The extracellular concentration dependence of amiloride block yields a Hill coefficient equal to 2, inconsistent with a single site blocking stoichiometry. 5. The shapes of current-voltage relationships measured in different external amiloride concentrations also indicate deviations from a simple channel plug model in which a single blocking cation is driven into the channel by the membrane potential. 6. Internal amiloride causes a voltage-independent 'flickery' block of mechanosensitive channel currents which equally reduces both inward and outward mechanosensitive currents. 7. The present data indicate that a minimum of two amiloride binding sites are necessary to predict external amiloride block. A model involving a voltage-dependent conformational change with subsequent voltage-independent co-operative binding of two amiloride molecules is found to explain the data. 8. The relevance of the present actions of amiloride on mechanosensitive channels is discussed in relation to reports of amiloride-inhibitable cation flux pathways involved in a number of basic physiological functions including mechanosensitivity of sensory cells, volume regulation and fertilization.

Amiloride↗