Owen P. Hamill

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches

Abstract1.The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches.2.A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipettemembrane seals with resistances of 109–1011Ω.3.The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels.4.Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals.5.A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution.6.The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (<20μm).7.The wide range of cell types amenable to giga-seal formation is discussed.

Mechanism of anion permeation through channels gated by glycine and gamma‐aminobutyric acid in mouse cultured spinal neurones.

1. The ion‐selective and ion transport properties of glycine receptor (GlyR) and gamma‐aminobutyric acid receptor (GABAR) channels in the soma membrane of mouse spinal cord neurones were investigated using the whole‐cell, cell‐attached and outside‐out patch versions of the patch‐clamp technique. 2. Current‐voltage (I‐V) relations of transmitter‐activated currents obtained from whole‐cell measurements with 145 mM‐Cl‐ intracellularly and extracellularly, showed outward rectification. In voltage‐jump experiments, the instantaneous I‐V relations were linear, and the steady‐state I‐V relations were rectifying outwardly indicating that the gating of GlyR and GABAR channels is voltage sensitive. 3. The reversal potential of whole‐cell currents shifted 56 mV per tenfold change in internal Cl‐ activity indicating activation of Cl(‐)‐selective channels. The permeability ratio of K+ to Cl‐ (PK/PCl) was smaller than 0.05 for both channels. 4. The permeability sequence for large polyatomic anions was formate greater than bicarbonate greater than acetate greater than phosphate greater than propionate for GABAR channels; phosphate and propionate were not measurably permeant in GlyR channels. This indicates that open GlyR and GABAR channels have effective pore diameters of 5.2 and 5.6 A, respectively. The sequence of relative permeabilities for small anions was SCN‐ greater than I‐ greater than Br‐ greater than Cl‐ greater than F‐ for both channels. 5. GlyR and GABAR channels are multi‐conductance‐state channels. In cell‐attached patches the single‐channel slope conductances close to 0 mV membrane potential were 29, 18 and 10 pS for glycine, and 28, 17 and 10 pS for GABA‐activated channels. The most frequently observed (main) conductance states were 29 and 17 pS for the GlyR and GABAR channel, respectively. 6. In outside‐out patches with equal extracellular and intracellular concentrations of 145 mM‐Cl‐, the conductance states were 46, 30, 20 and 12 pS for GlyR channels and 44, 30, 19 and 12 pS for GABAR channels. The most frequently occurring main state was 46 pS for the GlyR and 30 pS for the GABAR channel. 7. Single‐channel conductances measured in equal 140 mM concentrations of small anions on both membrane faces revealed a conductance sequence of Cl‐ greater than Br‐ greater than I‐ greater than SCN‐ greater than F‐ for both channels. This is nearly the inverse sequence of that found for the permeability of these ions indicating the presence of binding sites for ions in the channel.(ABSTRACT TRUNCATED AT 400 WORDS)

TRPC1 forms the stretch-activated cation channel in vertebrate cells

The mechanosensitive cation channel (MscCa) transduces membrane stretch into cation (Na+, K+, Ca2+ and Mg2+) flux across the cell membrane, and is implicated in cell-volume regulation, cell locomotion, muscle dystrophy and cardiac arrhythmias. However, the membrane protein(s) that form the MscCa in vertebrates remain unknown. Here, we use an identification strategy that is based on detergent solubilization of frog oocyte membrane proteins, followed by liposome reconstitution and evaluation by patch-clamp. The oocyte was chosen because it expresses the prototypical MscCa (≥107MscCa/oocyte) that is preserved in cytoskeleton-deficient membrane vesicles. We identified a membrane-protein fraction that reconstituted high MscCa activity and showed an abundance of a protein that had a relative molecular mass of 80,000 (Mr 80K). This protein was identified, by immunological techniques, as the canonical transient receptor potential channel 1 (TRPC1). Heterologous expression of the human TRPC1 resulted in a >1,000% increase in MscCa patch density, whereas injection of a TRPC1-specific antisense RNA abolished endogenous MscCa activity. Transfection of human TRPC1 into CHO-K1 cells also significantly increased MscCa expression. These observations indicate that TRPC1 is a component of the vertebrate MscCa, which is gated by tension developed in the lipid bilayer, as is the case in various prokaryotic mechanosensitive (Ms) channels.

Revisiting TRPC1 and TRPC6 mechanosensitivity

This article addresses whether TRPC1 or TRPC6 is an essential component of a mammalian stretch-activated mechano-sensitive Ca2+ permeable cation channel (MscCa). We have transiently expressed TRPC1 and TRPC6 in African green monkey kidney (COS) or Chinese hamster ovary (CHO) cells and monitored the activity of the stretch-activated channels using a fast pressure clamp system. Although both TRPC1 and TRPC6 are highly expressed at the protein level, the amplitude of the mechano-sensitive current is not significantly altered by overexpression of these subunits. In conclusion, although several TRPC channel members, including TRPC1 and TRPC6, have been recently proposed to form MscCa in vertebrate cells, the functional expression of these TRPC subunits in heterologous systems remains problematic.

Twenty odd years of stretch-sensitive channels

After formation of the giga-seal, the membrane patch can be stimulated by hydrostatic or osmotic pressure gradients applied across the patch. This feature led to the discovery of stretch-sensitive or mechanosensitive (MS) channels, which are now known to be ubiquitously expressed in cells representative of all the living kingdoms. In addition to mechanosensation, MS channels have been implicated in many basic cell functions, including regulation of cell volume, shape, and motility. The successful cloning, overexpression, and crystallization of bacterial MS channel proteins combined with patch clamp and modeling studies have provided atomic insight into the working of these nanomachines. In particular, studies of MS channels have revealed new understanding of how the lipid bilayer modulates membrane protein function. Three major membrane protein families, transient receptor potential, 2 pore domain K+, and the epithelial Na+ channels, have been shown to form MS channels in animal cells, and their polymodal activation embrace fields far beyond mechanosensitivity. The discovery of new drugs highly selective for MS channels (“mechanopharmaceutics”) and the demonstration of MS channel involvement in several major human diseases (“mechanochannelopathies”) provide added motivation for devising new techniques and approaches for studying MS channels.

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 ∼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 Ca2+ 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.

Gramicidin A channels switch between stretch activation and stretch inactivation depending on bilayer thickness

The patch clamp-liposome technique was used to examine the stretch sensitivity of a model membrane ion channel, gramicidin A, in membrane patches of different bilayer thickness. We found that small changes in phospholipid acyl chain length (i.e., PC-20 to PC-18) can switch gramicidin A from a stretch-activated to a stretch-inactivated channel. The demonstration that subnanometer changes in bilayer thickness can reverse the response polarity of a model channel has implications for other signaling proteins that may experience local changes in bilayer thickness as a consequence of dynamic targeting to lipid microdomains, electrocompression, or chemical modification of the bilayer.

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.

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

A novel technique involving hypertonic stress causes membrane ‘blebbing’ of the Xenopus oocyte and the shedding of plasma membrane vesicles (PMVs). 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. 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. 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. 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. 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.

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.