Specificity of ion channel inhibitors for the maxi cation channel in rye root plasma membranes

Journal of Experimental Botany, May 1996

The pharmacology of the maxi cation channel in the plasma membrane of rye (Secale cereale L.) root cells was studied following its incorporation into planar lipid bilayers. The channel was inhibited by ruthenium red, diltiazem, verapamil, and quinine at micromolar concentrations and TEA+ at millimolar concentrations.

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Specificity of ion channel inhibitors for the maxi cation channel in rye root plasma membranes

Philip J. White 0 0 Department of Cell Physiology, Horticulture Research International , Wellesbourne, Warwick CV35 9EF, UK The pharmacology of the maxi cation channel in the plasma membrane of rye [Secale cereale L.) root cells was studied following its incorporation into planar lipid bilayers. The channel was inhibited by ruthenium red, diltiazem, verapamil, and quinine at micromolar concentrations and TEA+ at millimolar concentrations. Introduction Pharmaceuticals that interact with ion channels specifically, and with high-affinity, can be applied both in vivo, to elucidate the role of an ion channel (and the flux of ions through it) in a particular physiological process, and in vitro, to label channel proteins during their purification or to provide an affinity-matrix for channel-protein purification. Thus, the screening for pharmaceuticals that interact with ion channel proteins is a prerequisite for certain studies of their physiology and biochemistry. Recently, the cation permeation and gating kinetics of a high conductance (maxi) cation channel in the plasma membrane of rye roots were described (White, 1993a). This channel is permeable to a wide variety of monovalent and divalent cations. However, under physiological ionic conditions, it appears to be activated by plasma membrane depolarization and to mediate both Ca2+ influx into the root cell and net K+ efllux (White, 1993a). It has been argued that the channel may be involved in intracellular signalling. It will open in response to a stimulus which depolarizes the plasma membrane, contributing to a rise in cytoplasmic Ca2+ concentration and the initiation of a physiological response. In this paper the effects of a range of pharmaceuticals on the activity Plasma-membrane vesicles were obtained by aqueous-polymer two-phase partitioning of a microsomal fraction from rye roots. The procedure followed White and Tester (1992) except that the homogenization medium additionally included 2 mM phenylmethylsulphonylfluoride, 4 mM dithioerythritol and 0.5% (w/v) polyvinylpyrrolidone as protectants. Plasma membrane vesicles were resuspended at a concentration of 1 mg protein ml"1 in 5 mM JV-/rts-[hydroxymethyl]-methyl-2-aminoethane sulphonic acid (TES), titrated to pH 7.5 using N-methylD-glucamine (NMDG) and stored at -20 C. Ion channel recordings Electrical recordings of ion channel activity were obtained following the incorporation of plasma membrane vesicles into planar lipid bilayers (PLB) composed of 30 mM synthetic 1 -palmitoyl-2-oleoyl phosphatidylethanolamine dispersed in ndecane, as described by White and Tester (1992). The bilayer (0.2 mm in diameter) separated solutions of 500 /A contained within a styrene copolymer cup (cis chamber) and 5 ml in an outer Perspex chamber (trans chamber). Aqueous solutions were filtered (pore diameter 0.2 ^m) and buffered with 5 mM TES, titrated to pH 7.5 using NMDG. Plasma membrane vesicles were added to the cis chamber and fused to bilayers by stirring in the presence of a (cis~.trans) 410:100 mM ICQ gradient. When channel activity was detected, unfused vesicles were removed by perfusing the cis chamber with 100 mM KC1. Pharmaceuticals were added to the cis or trans chamber from stock solutions. Diltiazem and ruthenium red were added from aqueous stocks of lOmM and 5 mM, respectively. Nifedipine was added from a 100 mM stock in acetone and verapamil was added from a 100 mM stock in ethanol. Stock solutions of tetraethylammonium chloride (TEAC1, 100 mM and 3 M) and quinine (1 mM) were made up in 100 mM KG and 5 mM TES titrated to pH 7.5 with NMDG. To obtain quinine concentrations above 100 ^M, quinine was added from a 100 mM stock in ethanol. The KC1 concentration was maintained at 100 mM by addition of KC1 from a 3 M stock solution made up in 5 mM TES titrated to pH 7.5 with NMDG when appropriate. Experiments were performed at room temperature (17-23C). Current was monitored under voltage-clamp conditions using a low noise operational amplifier with frequency compensation, connected to the bilayer chambers by calomel electrodes and 3 M KC1 salt bridges. Data were stored on digital audio tape (DTC1000ES; 44.1 kHz per channel; Sony Corporation, Japan). Following the bilayer convention, membrane potentials were recorded cis with respect to trans, which was held at ground. Since plasma membrane ion channels become oriented with their cytoplasmic face exposed to the trans chamber the sign of the membrane potential is opposite to that conventionally used in electrophysiological experiments of plant cells in vivo (White and Tester, 1994). For the illustration, single channel recordings were replayed, filtered at 1 kHz using an 8-pole low pass Bessel filter (902LPF, Frequency Devices Inc., Haverhill, Massachusetts, USA) and plotted directly using a DC signal conditioner (Model 13-6615-10A) and thermal chart recorder (Model TA420S) from Gould Electronics (Hainault, Essex, UK). Results and discussion The action of (...truncated)


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Philip J. White. Specificity of ion channel inhibitors for the maxi cation channel in rye root plasma membranes, Journal of Experimental Botany, 1996, pp. 713-716, 47/5, DOI: 10.1093/jxb/47.5.713