Abstract
A K+ channel was incorporated into voltage-clamped planar lipid bilayers from bovine chromaffin granules and resealed granule membranes (“ghosts”). It was not incorporated from plasma membrane-rich fractions from the adrenal medulla. The channel had a conductance of ∼ 400 pS in symmetric 450 mM KCI, with the permeability sequence K+ > Rb+ > Cs+ > Na+ > Li+, and was insensitive to both Ca2+ and charybdotoxin. It exhibited complex gating kinetics, consistent with the presence of multiple open and closed states, and its gating was voltage-dependent. The channels appeared to incorporate into bilayers with the same orientation, and were blocked from one side (the side of vesicle addition) by 0.2-1 mM TEA'. The block was slightly voltage-dependent. Acidification of resealed granule membranes in response to external ATP (which activated the vacuolartype ATPase) was significantly reduced in the presence of 1 mM intralumenal TEACI (with 9 mM KCl), and parallel measurements with the potential-sensitive dye Oxonol V showed that such vesicles tended to develop higher internal-positive membrane potentials than control vesicles containing only 10 mM KCI. 1 mM TEA+ had no effect on proton-pumping activity when applied externally, and did not directly affect either the proton-pumping or ATP hydrolytic activity of the partially-purified ATPase. These results suggest that chromaffin granule membranes contain a TEA+-sensitive K+ channel which may have a role in regulating the vesicle membrane potential.
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Apps DK, Pryde JG, Sutton R, Phillips JH (1980) Inhibition of adenosine triphosphatase, 5-hydroxytryptamine transport and proton-translocation activities of resealed chromaffin granule “ghosts”. Biochem J 190:273–282
Arispe N, Pollard H, Rojas E (1992) Calcium-independent K+-selective channel from chromaffrn granule membranes. J Membr Biol 130:191–202
Ashley RH (1989) Activation and conductance properties of ryanodine-sensitive calcium channels reconstituted from brain microsomal membranes. J Membr Biol 111:179–189
Bennett AB, Spanswick RM (1983) Optical measurement of ΔpH and Δψ on corn root membrane vesicles: kinetic analysis of C− effects on a proton-translocating ATPase. J Membr Biol 71:75–107
Bowman EJ, Siebers A, Altendorf K (1988) Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells and plant cells. Proc Natl Acad Sci USA 85:7972–7976
Casadio R (1991) Measurement of transmembrane pH differences of low extents in bacterial chromatophores. Eur J Biophys 19:189–201
Coronado R, Miller C (1979) Voltage-dependent cesium blockade of a cation channel from fragmented sarcoplasmic reticulum. Nature 280:807–810
Cukierman S, Yellen G, Miller C (1985) The K+ channel of sarcoplasmic reticulum. A new look at Cs+ block. Biophys J 48:477–484
Forgac M (1989) Structure and function of the vacuolar class of ATP-driven proton pumps. Physiol Rev 69:765–796
Haigh JR, Parris R, Phillips JH (1989) Free concentrations of sodium, potassium and calcium in chromaffin granules. Biochem J 259:485–491
Hayman KA, Spurway TD, Ashley RH (1993) Single anion channels reconstituted from cardiac mitoplasts. J Membr Biol 136:181–190
Hermann A, Gorman ALF (1981) Effects of tetraethylammonium on potassium currents in a molluscan neuron. J Gen Physiol 78:87–110
Hille B (1984) Ionic Channels of Excitable Membranes, First Edition. Sinauer Associates, Sunderland, MA
Hille B, Schwarz W (1978) Potassium channels as multi-ion single file pores. J Gen Physiol 72:409–442
Holz RW (1979) Measurement of membrane potential of chromaffin granules by reaccumulation of triphenylmethylphosphonium cation. J Biol Chem 254:6703–6709
Jan LY, Jan YN (1992) Structural elements involved in specific K+ channel functions. Annu Rev Physiol 54:537–555
Johnson RG (1988) Accumulation of biological amines into chromaffin granules: a model for hormone and neurotransmitter transport. Physiol Rev 68:232–307
Johnson RG, Scarpa A (1979) Protonmotive force and catecholamine transport in isolated chromaffin granules. J Biol Chem 254:3750–3760
Keller BU, Hedrich R (1992) Patch clamp techniques to study ion channels from organelles. Methods Enzymol 207:673–681
Latorre R, Vergara C, Moczydlowski E (1983) Properties of a Ca+-activated K+ channel in a reconstituted system. Cell Ca 4:343–357
Markwell MA, Haas SM, Tolbert NE, Bieber LL (1981) Protein determination in membrane and lipoprotein samples: manual and automated procedures. Methods Enzymol 72:296–303
Marty A (1981) Ca-dependent K channels with large unitary conductance in chromaffin cell membranes. Nature 291:497–500
Marty A, Neher E (1985) Potassium channels in cultured bovine adrenal chromaffin cells. J Physiol (London) 367:117–141
Meyer DI, Burger MM (1979) Isolation of a protein from the plasma membrane of adrenal cells which binds to secretory vesicles. J Biol Chem 254:1979–1987
Miller C, Moczydlowski E, Latorre R, Phillips M (1985) Charybdotoxin, a protein inhibitor of single Ca2+-activated K+ channels from mammalian skeletal muscle. Nature 313:316–318
Monck JR, Fernandez JM (1992) The exocytotic fusion pore. J Cell Biol 119:1395–1404
Moriyama Y, Nelson N (1987) The purified ATPase from chromaffin granule membranes is an anion-dependent proton pump. J Biol Chem 262:9175–9180
Njus D, Kelley PM, Harnadek GJ (1986) Bioenergetics of secretory vesicles. Biochim Biophys Acta 853:237–265
Ornberg RL, Kuijpers GAJ, Leapmean RD (1988) Electron probe microanalysis of the subcellular compartments of bovine adrenal chromaffin cells. J Biol Chem 263:1488–1493
Percy JM, Pryde JG, Apps DK (1985) Isolation of ATPase I, the proton pump of chromaffin granule membranes. Biochem J 231:557–564
Pérez-Castiñeira JR, Apps DK (1990) Vacuolar H+-ATPase of adrenal secretory granules. Rapid partial purification and reconstitution into proteoliposomes. Biochem J 271:127–131
Peterson GL (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83:46–356
Phillips JH, Apps DK (1980) Stoichiometry of catecholamine/proton exchange across the chromaffin granule membrane. Biochem J 192:273–278
Picaud S, Marty A, Trautmann A, Grynszpan-Winograd O, Henri J-P (1984) Incorporation of chromaffin granule membranes into large-size vesicles suitable for path-clamp recording. FEBS Lett 178:20–24
Rahamimoff R, DeRiemer SA, Sakmann B, Stadler H, Yakir N (1988) Ion channels in synaptic vesicles from Torpedo electric organ. Proc Natl Acad Sci USA 85:5310–5314
Robitaille R, Garcia ML, Kaczorowski GJ, Charlton MP (1993) Functional colocalization of calcium-gated potassium channels in control of transmitter release. Neuron 11:645–655
Sato M, Inoue K, Kasai M (1992) Ion channels on synaptic vesicle membranes studied by planar lipid bilayer method. Biophys J 63:1500–1505
Schuldiner S, Rottenberg H, Avron M (1972) Determination of ΔpH in chloroplasts. 2. Fluorescent amines as a probe for the determination of ΔpH in chloroplasts. Eur J Biochem 25:64–70
Stanley EF, Ehrenstein G, Russell JT (1988) Evidence for anion channels in secretory vesicles. Neuroscience 25:1035–1039
Thomas L, Hartung K, Langosch D, Rehm H, Bamberg E, Franke WW Betz H (1988) Identification of synaptophysin as a hexameric channel protein of the synaptic vesicle membrane. Science 242:1050–1053
Woodhull AM (1973) Ionic blockage of sodium channels in nerve. J Gen Physiol 61:687–708
Yellen G (1984) Ionic permeation and blockade in Ca+-activated K+ channels of bovine chromaffin cells. J Gen Physiol 84:157–186
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Correspondence to: R. H. Ashley
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Ashley, R.H., Brown, D.M., Apps, D.K. et al. Evidence for a K+ channel in bovine chromaffin granule membranes: single-channel properties and possible bioenergetic significance. Eur Biophys J 23, 263–275 (1994). https://doi.org/10.1007/BF00213576
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DOI: https://doi.org/10.1007/BF00213576