Vol. 41, No. 4, April 1997
BIOCHEMISTRYend MOLECULAR BIOLOGY INTERNATIONAL Pages 679-686
EFFECTS OF K* CHANNEL INHIBITORS ON POTASSIUM TRANSPORT IN BOVINE ADRENAL CHROMAFFIN GRANULES Nikolai A. Lobanov ~ , A d a m Szewczyk .2, Gra:,yna W6jcik 2, Marcin Nowotny 2 and Maciej J. Nal~cz 2 1Institute of Bioorganic Chemistry, Belorussian Academy of Sciences, Zhodinskaya 5/2, Minsk, 220141, Belarus; 2Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland Received January 6, 1997
SUMMARY
The Ca 2§ - independent W selective channel in the membrane of adrenal gland chromaffin granules has earlier been identified. In the present report we describe new properties of this potassium channel using 8~Rb§ a t C analogue, flux measurements. The studies are performed in membrane vesicles prepared from chromaffin granules. The electrogenic ~Rb + transport is inhibited by quinacrine, barium, zinc and magnesium. The effects of other potassium channel blockers on 86Rb§ transport into chromaffin granules are also reported.
Key Words: potassium transport, quinacrine, chromaffin granules, adrenal glands
INTRODUCTION
Recently, the Caz§ independent K§ selective channel (K+cG channel) of large conductance (150-300 pS) in the membrane of adrenal gland chromaffin granules has been described (1, 2). The K+cG channel is investigated after the fusion of purified granules membranes with planar bilayer membranes, followed by single channel recordings (1,2). Both intact chromaffin granules and purified granules membrane are fused with planar bilayer membranes and revealed the presence of the tCcc selective Abbreviations: K+CG - the Ca 2§ - independent K§ selective channel of adrenal gland chromaffin granules; TEA - tetraethylamonium cation; SMP - submitochondrial particles; TMB-8 - 8-(N, N-diethylamino)octyl-3, 4, 5 - trimethoxybenzoate). *Corresponding author, email:
[email protected] 1039-9712/97/040679-08505.00/0 679
C~q~yright 9 1997 by Academic Press Australia. All rights ~f reproduction in any,form reserved.
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL
Vol. 41, No. 4, 1997
channel, that is insensitive to the electrical potential and to Ca 2§ or charybdotoxin - a blocker of a CaZ*-activated K+ channel of large conductance (1). Sensitivity of the K+cG channel to TEA is also shown (2). Recently, it is reported that the WcG channel is controlled by both inhibitory and stimulatory heterotrimeric GTP-binding proteins (3). One problem regarding ionic channel studies in planar bilayer membranes is the purity of the applied biological membrane preparation. Therefore, we try to use flux measurements (4, 5) of 86Rb,+ a granule
K + channel
K+
analog, in order to study properties of the chromaffin
since this method
allows
specific measurements
in an
heterogeneous population of vesicles. Experiments are performed under conditions where only electrogenic transport of 8eRb+ is measured. This simple and convenient flux assay, combined with marker enzyme estimations, gives a valuable measuring of the K+CGchannel activity. Through this approach, we are able to present new properties of electrogenic K+ transport in chromaffin granules. This paper shows the effect of divalent cations and other potassium channel inhibitors on the electrogenic K+ transport in chromaffin granules vesicles. MATERIALS AND METHODS Materials - 8eRbCI, with a specific radioactivity of 20 Ci/mmol, was purchased from
Polatom (Poland) or from Amersham (UK). All other chemicals were of the highest purity commercially available. Subcellular fractionation of adrenal .qlands Bovine adrenal medullae are fractionated as described in (6) leading to microsomal, mitochondrial and the crude chromaffin granule preparations. Submitochondrial particles (SMP) are obtained as previously described (7). The crude chromaffin granule pellet is resuspended in the homogenization buffer, overlaid on 1.7 M sucrose, 1 mM EGTA, 1 mM MgSO4, 5 mM Hepes, pH 7.3, and centrifuged at 100000 x g for 60 min at 4~ The granular pellet is resuspended in 25 volumes of solution containing lmM MgSO4, 20mM Hepes, pH 7.3 and lysed by freezing and thawing. Subsequently, the suspension of chromaffin granule membranes is centrifuged at 48000 x g for 30 min. The obtained pellet is resuspended in the lysis buffer, and the centrifugation is repeated. Further purification is carried out by repeating resuspension of the pellet, its placing over a 1 M sucrose cushion and centrifugation. In detail, membrane suspension (2.5 ml) is overlaid on top of 12 ml of solution containing 1 M sucrose, 10 mM Hepes/KOH, pH 7.3 in Beckman SW28.1 rotor tubes and centrifuged at 100000 x g for 90 min. The chromaffin granule membranes remaining on top of the sucrose cushion are collected and resuspended in 25 volumes of 10 mM Hepes/KOH buffer, pH 7.4. These membranes are then centrifuged at 48000 x g for 30 min and, subsequently, the pellet is resuspended in the buffer containing 100 mM KCl, 10 mM Hepes/Tris, pH 7.4 at a protein concentration of 1 mg/ml. Aliquots of this final suspension are frozen. 680
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BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL
86Rb* flux measurements - Isotope fluxes through ion-conducting pathways are performed essentially as described before (4, 5). In brief, for transport experiments with membrane preparations the external K* is removed by passing the vesicles through a cation-exchange column (Dowex 50-X8 Tris form). Aliquots of 100 pl of the vesicle (chromaffin granule ghosts, SMP or microsomes) suspension are applied to small Dowex columns and eluted with 1.5 ml of 175 mM sucrose. This step is performed to exchange the external potassium cations into Tris § and to dilute the suspension approx. 5 fold. Various reagents are added, as required, to the vesicle suspension. The assay is initiated 30 s later by adding 25-50 ILl of 8eRbCI (2-4 #Ci). Vesicles are incubated with the isotope under different conditions and for the times indicated in figure legends. Subsequently, in order to separate the vesicles from the medium, 100 ILl aliquots of the reaction mixture are applied to 2-3 cm columns of Dowex 50-X8 (Tris form) placed in Pasteur pipettes, and vesicles are eluted directly into the counting vials by addition of 1.5 ml of ice-cold sucrose solution (175 mM). Prior to use, the columns are washed with 2 ml of 175 mM sucrose and then with 2 ml of 175 mM sucrose containing 25 mg/ml of bovine serum albumin and, stored at 4~ The amount of 86Rb§ trapped within the vesicles (eluted on the Dowex columns) is estimated by scintillation counting. The 86Rb+ content is expressed as a percentage of the initial total radioactivity in the vesicle reaction medium, or as a percentage of the control (sample without added reagents). Marker enzyme activity measurements. 5'-Nucleotidase and glucose-6-phosphatase are assayed by measuring the release of inorganic phosphate from AMP and from glucose-6-phosphate, respectively (8). The assay of inorganic phosphate is based on the method of Fiske and SubbaRow (9). Cytochrome c oxidase activity, the mitochondrial inner membrane marker enzyme, is measured as described in (10). The presence of a chromaffin granule membrane marker, cytochrome bsel, is measured by difference spectrum between dithionite-reduced and oxidized states at 561 nm (11 ). Protein concentration assay - Protein concentration is measured using the Bio-Rad Protein Assay kit according to the instructions of the manufacturer. RESULTS A N D DISCUSSION
The principle of measurements applied in the current studies is originally described by other investigators (4, 5). The chromaffin vesicles are prepared to contain 100 mM KCI inside. Shortly before starting the assay, the external 100 mM K+ is replaced with Tris § ion on DOWEX ion-exchange resin that results in the creation of a K+ gradient and, subsequently, in the efflux of K+ from chromaffin granule vesicles. An electrical diffusion potential (negative inside) is established in vesicles containing active K§ channels. Thus, an addition of 86Rb*, a K § analog, to the external solution should lead to the uptake of 8eRb+, against the K* gradient, but exclusively in the vesicles containing active channels. Such a process is possible due to the e6Rb§ equilibration according to the diffusion potential previously built up across the granular membrane. This
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phenomena amplifies the sensitivity of transport measurements. In addition, the 8eRb* uptake does not affect the potential itself. An application of this method has essential advantages for studies of intracellular ion channels. Firstly, procedures of the tissue fractionation very often lead to the production of a heterogeneous vesicle population, creating difficulties in studying the single channel properties in black lipid membranes. Instead, flux studies with membrane vesicles, plus estimation of suitable marker enzymes, allow us to correlate the measured transport activity with a specific intracellular compartment. Secondly, flux measurements enable studies of numerous ionic channel effectors within a short time. In order to verify whether the measured S6Rb+ uptake is localized in chromaffin granules membranes, both transport activity and maker enzyme level are estimated in different
populations
of vesicles
obtained during
adrenal
gland
fractionation.
Identification of a proper fraction is found to be especially important in the light of the ubiquitous presence of potassium channels in the intracellular membranes
of
endoplasmic reticulum and mitochondria (for review see [12, 13]). Figure 1 shows the comparison of 86Rb+ uptake with the level of cytochrome b~el, the marker enzyme of
0.015
z
