Single acetylcholine receptor channel currents recorded at high hydrostatic pressures.

Abstract

A technique for performing patch-clamp experiments under high hydrostatic (oil) pressure is described. The method allows the transfer of whole cells or membrane patches in a recording configuration into a pressure vessel, where pressure can be increased up to 60 MPa (= 600 bar). We have studied in this way the pressure dependence of single acetylcholine receptor channels in excised "outside-out" membrane patches from cultured rat muscle cells. In the range of 0.1 to 60 MPa the open channel conductance in 140 mM NaCl solutions did not vary by more than 2%, which implies that the translocation of sodium ions through the channel pore does not involve steps with significant activation volumes. The activation volumes for the gating of acetylcholine receptor channels are comparable to those of sodium and potassium channels in the squid giant axon, suggesting that there is some basic common mechanism in the operation of ion-channel proteins.Hydrostatic pressure is a standard tool for studying chemical reactions, because reaction rates and equilibria can be strongly dependent on this thermodynamic parameter (1). The effects of pressure on biological molecules (2) must be ultimately responsible for the profound physiological effects of hyperbaric conditions on living organisms and isolated tissues (3, 4). Pressure-dependent properties have been already observed for the proteins providing the sodium and potassium pathways in squid giant axons and snail neurons (5-8) and for the pores formed by alamethicin in artificial bilayers (9). We describe here an extension of the patchclamp technique (10-12) that allows patch-clamp recordings at high hydrostatic pressures. This method gives access to a broad field of physiological and biophysical investigations on the effects of pressure on ion channels in small mammalian cells. As a first example of such an application we report here studies on the pressure dependence of the nicotinic acetylcholine receptor (AcChoR) channels in isolated membrane patches from cultured muscle cells.The AcChoR channels are well characterized under atmospheric conditions (10-16). They mediate the transmission of electrical signals across the synaptic cleft, so that the pressure dependence of their properties might play a major role in the complex phenomenology underlying the "high pressure nervous syndrome." Under normal conditions the ion flow through the AcChoR pore yields an electrical current on the order of 2 pA lasting for most events between 5 and 50 ms. These values define the minimal resolution required from the electrical recording system. MATERIALS AND METHODSCell Preparation. Our studies were carried out on cultured embryonic muscle cells of rat. Muscles of newborn Wistar rats were dissociated and stored in culture. After 3 days the cells were treated with colchicine in order to destroy the cytoskeleton (13). Between day 4 and day 10 in culture the now-round myoballs could be used for the experiments.The ionic concentrations of the standard bath solution used in most experiments wer...

Proceedings of the Physiological Society, 14‐15 February 1992, Bristol Meeting: Communications

“…Recordings were made between 30 and 180 min after compression and were low pass filtered at 1 kHz and analysed off-line (Dempster, 1988 (Mj), open probability and frequency (PO and F) all showed a biphasic pattern of response. The results are not consistent with pressure primarily affecting the agonist-receptor interaction or mimicking the effect of cooling, and they are unlike the effects of hydrostatic pressure on the Ach-R channel (Heinemann et al 1987). (Silver et al 1990).…”

Neuronal sensitivity to hyperoxia, hypercapnia, and inert gases at hyperbaric pressures

“…The tissue preparation is submerged in a static bath, and a pneumatic pump delivers compression medium to the interior of the chamber, completely displacing any gas overlying the tissue bath. Cam-driven microdrives are used to manipulate the recording microelectrode, rather than electric microdrives, because the latter are incompatible with aqueous compression medium (91,166,215). Typically, adiabatic temperature changes occur during rapid compression and decompression that require several minutes to dissipate, which often confounds electrophysiological data collected during the act of compressing and decompressing the chamber (91,215).…”

“…The small internal volume of a hydrostatic compression chamber is beneficial because it enables the investigator to rapidly increase ambient pressure when studying the biophysical effects of an extreme level of hyperbaria (184). Hydrostatic compression chambers have typically been used to impose extremely high levels of hydrostatic pressure, usually Ͼ100 ATA, on nonmammalian neurons and axons (214,215) and large nonneuronal cells (91,184). The tissue preparation is submerged in a static bath, and a pneumatic pump delivers compression medium to the interior of the chamber, completely displacing any gas overlying the tissue bath.…”

“…Patch-clamp recordings of whole cell and single-channel ionic currents have been made at extremely high hydrostatic pressures in nonmammalian cells to study HPNS and the effects of large hyperbaric pressures (91,130,132,184), and during exposure to HBO 2 (133). This technique involves visualizing and patching onto a dissociated cell maintained in a removable bath that is attached to the stage of an inverted microscope.…”

“…Cam-driven microdrives are used to manipulate the recording microelectrode, rather than electric microdrives, because the latter are incompatible with aqueous compression medium (91,166,215). Typically, adiabatic temperature changes occur during rapid compression and decompression that require several minutes to dissipate, which often confounds electrophysiological data collected during the act of compressing and decompressing the chamber (91,215). In addition, because most chamber designs use a static bath, the composition of the tissue bath cannot be manipulated by the investigator, and the small internal volume of the chamber, which facilitates rapid compression and decompression, makes it difficult to position and manipulate the recording micropipette (184).…”