Specificity of ion channel inhibitors for the maxi cation channel in rye root plasma membranes
Philip J. White
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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
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