The heterogeneity of ion channels in chromaffin granule membranes

Cellular & Molecular Biology Letters, Sep 2006

Chromaffin granules are involved in catecholamine synthesis and traffic in the adrenal glands. The transporting membrane proteins of chromaffin granules play an important role in the ion homeostasis of these organelles. In this study, we characterized components of the electrogenic 86Rb+ flux observed in isolated chromaffin granules. In order to study single channel activity, chromaffin granules from the bovine adrenal medulla were incorporated into planar lipid bilayers. Four types of cationic channel were found, each with a different conductance. The unitary conductances of the potassium channels are 360 ± 10 pS, 220 ± 8 pS, 152 ± 8 pS and 13 ± 3 pS in a gradient of 450/150 mM KCl, pH 7.0. A multiconductance potassium channel with a conductivity of 110 ± 8 pS and 31 ± 4 pS was also found. With the exception of the 13 pS conductance channel, all are activated by depolarizing voltages. One type of chloride channel was also found. It has a unitary conductance of about 250 pS in a gradient of 500/150 mM KCl, pH 7.0.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

http://link.springer.com/content/pdf/10.2478%2Fs11658-006-0027-1.pdf

The heterogeneity of ion channels in chromaffin granule membranes

CELLULAR & MOLECULAR BIOLOGY LETTERS Volume 0 Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, Polish Academy of Sciences , Pasteur 3, 02-093 Warsaw , Poland 1 Department of Biophysics, Agriculture University SGGW , Nowoursynowska 159, 02-776 Warsaw , Poland Chromaffin granules are involved in catecholamine synthesis and traffic in the adrenal glands. The transporting membrane proteins of chromaffin granules play an important role in the ion homeostasis of these organelles. In this study, we characterized components of the electrogenic 86Rb+ flux observed in isolated chromaffin granules. In order to study single channel activity, chromaffin granules from the bovine adrenal medulla were incorporated into planar lipid bilayers. Four types of cationic channel were found, each with a different conductance. The unitary conductances of the potassium channels are 360 ± 10 pS, 220 ± 8 pS, 152 ± 8 pS and 13 ± 3 pS in a gradient of 450/150 mM KCl, pH 7.0. A multiconductance potassium channel with a conductivity of 110 ± 8 pS and 31 ± 4 pS was also found. With the exception of the 13 pS conductance channel, all are activated by depolarizing voltages. One type of chloride channel was also found. It has a unitary conductance of about 250 pS in a gradient of 500/150 mM KCl, pH 7.0. Chromaffin granule; Intracellular channel; Potassium channel; Chloride channel; Black lipid membrane THE HETEROGENEITY OF ION CHANNELS IN CHROMAFFIN GRANULE MEMBRANES Abbreviations used: BLM – black lipid membrane technique, I – single-channel current amplitude, KCG – large conductance potassium channel, Po – open-probability; U – potential, Urev – reversal potential, γ – ion conductance, τo – mean open lifetime, τc – mean closed lifetime Ion channels selective for potassium and chloride ions are present in all intracellular membranes [1]. They have been found in the endo/sarcoplasmic reticulum, Golgi apparatus, lysosomes, endosomes and mitochondria [2]. Recently, mitochondrial potassium channels have become a focus of researchers’ attention due to the observation that the pharmacological modulation [3, 4] of their activity yields a cytoprotective effect in different cell types [5, 6]. These channels are also present in the membranes of secretory vesicles such as chromaffin and zymogen granules, and synaptic vesicles [7]. Intracellular pathways for K+ and Cl- transport via ion channels are involved in a variety of processes, such as organelle volume regulation, charge compensation and pH homeostasis in specific cellular compartments [8-10]. Ion channels are also present in the membranes of chromaffin granules from the adrenal medulla. Chromaffin granules are involved in catecholamine synthesis and traffic. By the process of exocytosis, the chromaffin cell secretes a complex mixture of catecholamines, ATP, ions, proteins and peptides. The uptake of catecholamine into chromaffin granules is driven by the pH gradient [11]. The chromaffin granule ion channels were investigated via electrophysiological and macroscopic flux measurements. Single channel recordings were obtained both with the patch-clamp technique and after fusion of purified granule membranes with a bilayer membrane (BLM). Several different cation-selective channels were described after the incorporation of intact chromaffin granules, but only two types of highly selective K+ channel could be reconstituted from a preparation of chromaffin granule “ghosts” [12, 13]. A K+-selective, large conductance (~160 pS in symmetrical 400 mM KCl) channel was described in [12]. It was insensitive to charybdotoxin, a blocker of the Ca2+-activated K+ channel of large conductance. The channel’s activity was also unaffected by Ca2+ and potential across the bilayer [12]. It was also reported that the chromaffin granule K+ channel was controlled by both inhibitory and stimulatory heterotrimeric GTP-binding proteins [14]. A similar channel, highly selective for potassium, but with a different conductance (~400 pS in symmetric 450 mM KCl), was described in [13]. This channel was insensitive to both Ca2+ and + charybdotoxin, and was blocked by TEA . There is also evidence of anion channels in chromaffin granule membranes [15-17]. The application of 86Rb+ flux measurements to chromaffin granules proved the existence of a potent electrogenic, K+-selective transport system in the granular membrane. Using this technique, it was shown that K+ electrogenic transport is blocked by ATP and nucleotide analogues [18], by Mg2+, Ba2+ and Zn2+ [18, 19], and by SH-modifying agents such as N-ethylmaleimide or mersalyl [20]. We recently reported that 86Rb+ flux is blocked by low pH [21]. The aim of this study was to analyze the single channel components of the K+ electrogenic flux observed in chromaffin granule membranes. We characterized ion channels which may contribute to monovalent ion transport through chromaffin granule membranes. Channel activity was registered using the p (...truncated)


This is a preview of a remote PDF: http://link.springer.com/content/pdf/10.2478%2Fs11658-006-0027-1.pdf

Renata Hordejuk, Adam Szewczyk, Krzysztof Dołowy. The heterogeneity of ion channels in chromaffin granule membranes, Cellular & Molecular Biology Letters, 2006, pp. 312-325, Volume 11, Issue 3, DOI: 10.2478/s11658-006-0027-1