Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes

Abstract

High-affinity binding sites for m~no ['~~I]iodoapamin were detected in membranes (Kd = 59 pM, B,,, = 24 fmol/mg protein) and cultured cells (& = 69 pM, B,,, = 2.8 fmol/mg protein) from rat heart and in membranes from guinea-pig ileum (& = 67 pM, B, , , = 42 fmol/mg protein) and liver (Kd = 15 pM, B,,, = 43 fmol/ mg protein). Binding was stimulated by K t ions = 0.3-0.5 mM). Covalent labeling with arylazide ['251]iodoapamin derivatives showed that smooth muscle, liver and heart binding molecules are associated with a 85 -87-kDa polypeptide. A second strongly labeled 57-kDa component was identified in liver membranes only.Apamin, a bee-venom peptide, is a specific blocker of one type of CaZ +-activated K' channel. Although it was initially considered to be essentially a centrally acting neurotoxin, the first results demonstrating an effect of apamin in vitro were obtained in peripheral systems (for review see [l]) and its action on intestinal smooth muscle and hepatocyte suspensions has been well characterized using pharmacological and electrophysiological techniques [2 -71.Apamin can be radioiodinated to high specific activity and is a potentially useful ligand for a biochemical study of K + -channel-associated membrane binding sites. Work in this area has, however, been centered on the rat central nervous system receptor using covalent labeling, target-size analysis and detergent extraction and sedimentation analysis [8 -111. However, while high-affinity binding sites have also been described in guinea-pig but not rat hepatocytes [6] and in smooth muscle [7], no data on binding molecule structure in peripheral systems have, to our knowledge, been reported. In the present paper we describe photoaffinity labeling of the apamin receptor in plasma membrane fractions from guinea-pig ileum and liver. We also report the presence of apamin receptors in cultured rat cardiac cells and purified heart membranes. EXPERIMENTAL PROCEDURE Preparation of I-labeled 4-azido-2-nitrophenylaminoacetyl-apaminApamin was purified from bee venom [12], radioiodinated [ 131 and mono [' 251]iodo derivatives were separated by SP-(sulfopropy1)-Sephadex chromatography [14]. lz5I-1abeled 4-azido-2-nitrophenylaminoacetyl-apamin ('"I-ANPAAapamin) was prepared as previously described [9], in the presence of 1 mg/ml bovine serum albumin carrier protein, using a molar ratio of 4: 1 of azido-2-nitrophenylaminoacetyl- Membrane preparationSmooth muscle membranes were prepared from guineapig ileum by the method of Matlib et al. [l 51. After discontinuous density gradient centrifugation membranes from the 8 -30% sucrose interface were collected. In some experiments homogenization and preparation of the 100000 x g pellet were carried out in the presence of the protease inhibitors phenanthroline (1 mM) pepstatin A (1 pM), phenylmethylsulfonyl fluoride (0.1 mM) iodoacetamide (1 mM) and EDTA (2 mM) and the density-gradient step was omitted. Plasma membranes were prepared from guinea-pig liver following the procedure of Neville [16] up to step 11 and from ra...

Cloning of an apamin binding protein of vascular smooth muscle

“…1A) and the cross-linking experiments (Fig. 1B), our results do not support the suggestion, by others, that the smaller protein species ("p50 doublet" in this work) is also capable of binding apamin (Seagar et al, 1986;Marqueze et al, 1987).…”

Molecular characterization of volume-sensitive SKCachannels in human liver cell lines

“…In concentrations of 10-50 nM, apamin blocks the 42 K ϩ efflux and K ϩ conductance increase caused by adrenergic stimulation of isolated guinea pig hepatocytes (3,4) and prevents the increase in K ϩ conductance caused by metabolic stress in liver and biliary cell lines (22,23). Moreover, there appear to be ϳ1,700 apamin binding sites per liver cell, although more than one target protein may be involved (7,13). On the basis of these observations, the purpose of these studies was to assess whether apamin-sensitive SK Ca channels are expressed in liver epithelia and to evaluate whether they contribute to cell volume regulation and modulation of volume-sensitive liver functions.…”

Discrimination between subtypes of apamin‐sensitive Ca2+‐ activated K+ channels by gallamine and a novel bis‐quaternary quinolinium cyclophane, UCL 1530

“…Our tentative conclusions are however in keeping with other evidence that suggests that the SKCa channels are heterogeneous. Photoaffinity labelling and chemical crosslinking studies with [1251]-apamin have already identified a number of apamin binding polypeptides (Schmid-Antomarchi et al, 1984;Marqueze et al, 1987;Auguste et al, 1989), suggesting the SKCa channel is composed of several subunits. While all the cell types studied have yielded a protein of approximately 30 kDa, its size has been reported to range from 25 to 33 kDa, depending on the tissue.…”

“…In another approach, some progress has been made towards the biochemical characterization of apamin binding proteins-presumed to form part of the SKca channel (Schmid-Antomarchi et al, 1984; ' Author for correspondence. Marqueze et al, 1987;Auguste et al, 1989). An important recent study by Wadsworth et al (1994) has provided evidence that these proteins vary between species and in the same work it was shown that the neuromuscular blocking agent, gallamine, is less effective in displacing labelled apamin from its binding site in rat brain than in rabbit and guinea-pig liver.…”