Don W. McBride

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.

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 μM), gentamicin (220 μM), flufenamic acid (40 μM), niflumic acid (100 μM), Gd3+ (0.3 μM) or Ca2+ (200 μM) 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: a molecular probe for mechanosensitive channels

age, because CAMP mimics the glucagon effect on cell volume. On the other hand, when receptor- gated ion channels are activated by hormones, cell volume changes could represent a rather early event in the intracellular signal transduction. Remarkably, the cell volume response to Ca’+-mobilizing hormones is not uniform: phenyl- ephrine swells hepatocytes, whereas extracellular ATl? and vasopressin shrink theml’. This suggests that factors other than [Ca2+li are involved in cell volume changes under the influence of Ca’+-mobilizing hormones. In summary, currently available evidence suggests that changes in cell volume under the influence of hormones act as second or third messengers mediating some of the metabolic hormone effects. It is hoped that this article will stimu- late future research in this exciting area.

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 (≈ 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 muscle 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.

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 μm) 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 Ca2+‐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.

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 bromohexa‐methyleneamiloride (BrHMA) block the MS channel in a highly voltage‐dependent manner. 3 All analogues tested were more potent blockers than amiloride with IC50s of 500 μm (amiloride), 370 μm (DMA), 95 μm (benzamil) and 34 μm (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.

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.

A supramolecular complex underlying touch sensitivity.

Touch sensitivity in humans is dependent on highly specialized cutaneous nerve endings encapsulated in elaborate cellular structures such as the Pacinian, Ruffini and Meissners corpuscles. Although the details of the encapsulations vary, the common theme involves the nerve endings making intimate mechanical linkages with the collagen-fiber networks contained within each capsule1. Presumably, it is these external linkages with the membrane that serve to transmit and focus mechanical energy onto the mechanotransducers located in the nerve endings, and thus contribute to their low threshold and high mechanosensitivity.

A Fast Pressure-Clamp Technique for Studying Mechanogated Channels

Mechanosensitive (MS) membrane ion channels provide a means of transducing cell membrane deformation or stretch into an electrical or ionic signal (Howard et al.,1988; Sokabe and Sachs, 1992). They represent the most recently discovered and least understood of the major channel classes. It is only recently that information on their molecular nature has been provided (for references, see Hamill and McBride, 1994a). Yet MS channels are ubiquitous, being found in both eukaryotes and prokaryotes (Martinac, 1993). Although their role in mechanotransduction in sensory cells is evident, in nonsensory cells they have been implicated in diverse mechanosensitive functions (Sachs, 1988). There is a variety of MS channels with different gating (stretch-activated and stretch-inactivated) and ion-selective (Na+/K+, K+, Cl−, etc) properties (Morris, 1990). There also appear to be two broad mechanisms by which mechanosensitivity can be conferred on a channel. These are direct or indirect, according to the way mechanical energy is coupled to the gating mechanism. In direct coupling, mechanical energy acts directly on the channel molecule, and we refer to these as mechanogated (MG) channels. In indirect coupling the channel itself is not MS but is gated by a second messenger that is regulated by a MS enzyme or process (see Ordway et al., 1992).

Mechanogated Channels in Xenopus Oocytes: Different Gating Modes Enable a Channel to Switch From a Phasic to a Tonic Mechanotransducer

Critical to the survival of any cell is its ability to sense and respond appropriately to changes in its environment. In the case of the mechanical environment, there are both static and dynamic components that the cell may be required to selectively detect. Such detection takes place in the presence of a dynamic background of mechanical stimulation arising from Brownian motion, gravitational force, and various forces generated within a cell (e.g., due to molecular motors and cycles of cytoskeletal polymerization and depolymerization) that maintain cell shape and also mediate shape changes during growth and adhesion. In addition to such background forces, a cell may experience other mechanical perturbations, ranging from steady indentations to highfrequency vibrations, and from osmotic challenges to fluid shear stresses. Detection and appropriate responses to such perturbations may be critical for the function and, perhaps, survival of the cell. Therefore, cells require biological mechanotransducers that can extract specific information regarding relevant mechanical stimuli while filtering out irrelevant stimuli. A variety of mechanosensitive processes have been identified including (i) mechanosensitive enzymes such as adenylate cyclase (Watson, 1990) and phospholipase A2 (Jukka et al., 1995), (ii) mechanosensitive transmitter release (Chen and Grinnell, 1995), (iii) mechanosensitive gene activation (Sadoshima et al., 1992), and (iv) the