A look at membrane patches with a scanning force microscope

Hörber, J. K. H.; Mosbacher, Johannes; Häberle, W.; Ruppersberg, J. P.; Sakmann, Bert

doi:10.1016/s0006-3495(95)80346-2

Cited by 65 publications

(34 citation statements)

References 18 publications

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“…High-resolution images of membranes in patch pipettes have been presented previously (22,23,25,26), and it has been shown that rapid changes in voltage can cause rapid submicrometer movements of the suspended part of the membrane patch (27)(28)(29)(30). Our study shows that prolonged depolarization of a membrane patch in a borosilicate pipette can lead to substantial displacement of the membrane patch into the pipette and that this displacement correlates in time with the activation of MS channels in the same patch.…”

Section: Discussionmentioning

confidence: 53%

Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels

Gil

Silberberg

Magleby

1999

Proc. Natl. Acad. Sci. U.S.A.

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The patch-clamp technique allows currents to be recorded through single ion channels in patches of cell membrane in the tips of glass pipettes. When recording, voltage is typically applied across the membrane patch to drive ions through open channels and to probe the voltage-sensitivity of channel activity. In this study, we used video microscopy and single-channel recording to show that prolonged depolarization of a membrane patch in borosilicate pipettes results in delayed slow displacement of the membrane into the pipette and that this displacement is associated with the activation of mechanosensitive (MS) channels in the same patch. The membrane displacement, Ϸ1 m with each prolonged depolarization, occurs after variable delays ranging from tens of milliseconds to many seconds and is correlated in time with activation of MS channels. Increasing the voltage step shortens both the delay to membrane displacement and the delay to activation. Preventing depolarization-induced membrane displacement by applying positive pressure to the shank of the pipette or by coating the tips of the borosilicate pipettes with soft glass prevents the depolarization-induced activation of MS channels. The correlation between depolarization-induced membrane displacement and activation of MS channels indicates that the membrane displacement is associated with sufficient membrane tension to activate MS channels. Because membrane tension can modulate the activity of various ligand and voltage-activated ion channels as well as some transporters, an apparent voltage dependence of a channel or transporter in a membrane patch in a borosilicate pipette may result from voltage-induced tension rather than from direct modulation by voltage. M echanosensitive (MS) ion channels can act as transducers for the senses of touch, hearing, balance, and proprioception and also seem to be involved in the regulation of cell volume (1-3). MS channels in patches of membrane in the tips of patch pipettes are readily activated when the membrane is stretched by the application of negative pressure to the shank of the patch pipette (4). The stretching of the membrane patch by negative pressure is thought to activate the channels by increased tension in the membrane (5). When the pressure is released, the channels then readily deactivate. In addition to stretch activation, some MS channels can also be activated by voltage (1). In this regard, depolarization of membrane patches from Xenopus oocytes activates endogenous MS channels in the patch after prolonged delays of typically 1-20 s (6). After stepping the voltage back to negative potentials, the MS channels then deactivate over several seconds. The delayed activation is observed for both on-cell and excised patches and typically occurs in a cooperative manner, with all channels activating over a short period of time compared with the duration of the delay to activation.We have shown recently that the delayed activation of the MS channels is not observed in outside-out patches, in membrane outside of the patc...

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Section: Discussionmentioning

confidence: 53%

Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels

Gil

Silberberg

Magleby

1999

Proc. Natl. Acad. Sci. U.S.A.

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“…It may involve degradation of cytoskeleton elements 17 that are known to be included in the inside-out patch. 18 Although both oleoyl-CoA and PIP 2 rapidly activated K ATP channels after partial rundown, their rate of washout was very slow. After a 15-minute period of exposure to either of these molecules, almost no rundown of K ATP channels was observed during 30 minutes of washout with divalent-free solution ( Figure 1B, arrow).…”

Section: Resultsmentioning

confidence: 99%

Long-Chain Acyl–Coenzyme A Esters and Fatty Acids Directly Link Metabolism to K _ATP Channels in the Heart

Liu

Hanley

Ray

et al. 2001

Circulation Research

102

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Abstract-ATP-sensitive K (K ATP ) channels are inhibited by cytosolic ATP, a defining property that implicitly links these channels to cellular metabolism. Here we report a direct link between fatty acid metabolism and K ATP channels in cardiac muscle cells. Long-chain (LC) acyl-coenzyme A (CoA) esters are synthesized from fatty acids and serve as the principal metabolic substrates of the heart. We have studied the effects of LC acyl-CoA esters and LC fatty acids on K ATP channels of isolated guinea pig ventricular myocytes and compared them with the effects of phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Application of oleoyl-CoA (0.2 or 1 mol/L), a naturally occurring acyl-CoA ester, to the cytosolic side of excised patches completely prevented rundown of K ATP channels, but not of Kir2 channels. The open probability of K ATP channels measured in the presence of oleoyl-CoA or PIP 2 was voltage dependent, increasing with depolarization. Oleoyl-CoA greatly reduced the ATP sensitivity of K ATP channels. At a concentration of 2 mol/L, oleoyl-CoA increased the half-maximal inhibitory concentration of ATP Ͼ200-fold. The time course of the decrease in ATP sensitivity was much faster during application of oleoyl-CoA than during application of PIP 2 . The effects of PIP 2 , but not of oleoyl-CoA, were inhibited by increasing Ca 2ϩ to 1 mmol/L. Oleate (C18:1; 10 mol/L), the precursor of oleoyl-CoA, inhibited K ATP channels activated by oleoyl-CoA. Palmitoleoyl-CoA and palmitoleate (C16:1) exerted similar reciprocal effects. These findings indicate that LC fatty acids and their CoA-linked derivatives may be key physiological modulators of K ATP channel activity in the heart. (Circ Res. 2001;88:918-924.) Key Words: free fatty acids Ⅲ acyl-CoA esters Ⅲ PIP 2 Ⅲ K ATP channels A TP-sensitive potassium (K ATP ) channels link energy metabolism with the electrical activity of the heart. [1][2][3] This link is functionally important during hypoxia or ischemia. Any imbalance in the ratio between energy supply through coronary arteries and energy expenditure by cardiac myocytes will indirectly modulate electrical activity, because a change in the ATP/ADP ratio, together with acidosis, promotes activation of K ATP channels. 4 -6 By increasing K ϩ efflux and shortening the duration of the action potential, K ATP channel activation reduces transsarcolemmal Ca 2ϩ influx and thereby the energy costs of Ca 2ϩ -ATPases and actomyosin-ATPase. Hence, in the face of reduced energy supply, K ATP channels may provide a means to decrease cytosolic energy demand.We report here a direct effect of long-chain (LC) acylcoenzyme A (CoA) esters and fatty acids on the activity of K ATP channels in isolated cardiac myocytes. We have shown that LC acyl-CoA esters facilitate the opening of K ATP channels by reducing their ATP sensitivity. The precursors of LC acyl-CoA esters, LC fatty acids, were found to inhibit K ATP channels. The reciprocal effects of free fatty acids and acyl-CoA esters on K ATP channels represent a novel link between energy metab...

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“…In this regard, the regulation of myocardial KATP channels resembles the modulation of Nae and Cl-channels by actin filament disrupters, which can also proceed in excised membrane patches (Cantiello et al 1991;Prat et al 1995;Udrovinas, Shander & Makielski, 1995). This, however, does not necessarily mean that the action of actin filament disrupters could occur in a 'cytosol-free' environment since membrane patches do not consist of just the plasmalemma, but are composed of membrane-covered cytoplasm with cytoskeletal structures (Ruknudin et al 1991;Horber et al 1995). It has been proposed previously that actin, and actin A a ATP (200 1sM) binding proteins, are linked to various transport proteins including the ac-subunit of the Na+,K+-ATPase (Nelson & Veshmock, 1987), Na+ and Ca2+ channels in the brain (Srimivasan, Elmer, Davis, Bennett & Angelides, 1988;Johnson & Byerly, 1993), NMDA channels in the hippocampal neurons (Rosenmund & Westbrook, 1993), Nae and Cl channels in kidney (Cantiello et al 1991;Suzuki et al 1993) and the cystic fibrosis transmembrane conductance regulator (Prat et al 1995).…”

Section: Resultsmentioning

confidence: 99%

Actin microfilament disrupters enhance K(ATP) channel opening in patches from guinea‐pig cardiomyocytes.

Terzic

Kurachi

1996

The Journal of Physiology

101

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1. To determine whether actin filament networks are associated with the regulation of ATP‐sensitive K+ (K(ATP)) channel activity, single channel currents were measured in the inside‐out configuration, and cytoskeletal disrupters applied to the internal side of patches excised from guinea‐pig ventricular myocytes. 2. Treatment of patches with DNase I (10‐200 micrograms ml(‐1)), which forms complexes with G‐actin and prevents actin filament formation, antagonized the ATP‐induced inhibition of K(ATP) channels. 3. In the absence of ATP, DNase I did not increase K(ATP) channel activity. 4. When denatured by boiling or co‐incubated with purified actin subunits (200 mu g ml(‐1)), DNase I(100 mu g ml(‐1)) did not antagonize the ATP‐induced inhibition of K(ATP) channels. 5. The DNase I‐induced decrease in the sensitivity of K(ATP) channels towards ATP‐induced inhibition was partially restored by addition of purified actin subunits (200 micrograms ml(‐1)). 6. Cytochalasin B (10 microM), another actin filament disrupter, but neither taxol nor nocodazole (30‐100 microM), two antimicrotubule agents, enhanced K(ATP) channel activity in the presence of ATP. 7. Hence, actin filament disrupters can attenuate the ATP‐dependent inhibitory gating of K(ATP) channels. This suggests that subsarcolemmal actin filament networks may be associated with the regulation of cardiac K(ATP) channels.

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A look at membrane patches with a scanning force microscope

Cited by 65 publications

References 18 publications

Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels

Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels

Long-Chain Acyl–Coenzyme A Esters and Fatty Acids Directly Link Metabolism to K _ATP Channels in the Heart

Actin microfilament disrupters enhance K(ATP) channel opening in patches from guinea‐pig cardiomyocytes.

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A look at membrane patches with a scanning force microscope

Cited by 65 publications

References 18 publications

Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels

Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels

Long-Chain Acyl–Coenzyme A Esters and Fatty Acids Directly Link Metabolism to K ATP Channels in the Heart

Actin microfilament disrupters enhance K(ATP) channel opening in patches from guinea‐pig cardiomyocytes.

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Long-Chain Acyl–Coenzyme A Esters and Fatty Acids Directly Link Metabolism to K _ATP Channels in the Heart