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Solubilisation and reconstitution of the rabbit skeletal muscle sarcoplasmic reticulum K+ channel into liposomes suitable for patch clamp studies

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Abstract

Sarcoplasmic reticulum (SR) membrane vesicles have been prepared from rabbit skeletal muscle and solubilised using K+ cholate. Solubilised membrane proteins were reconstituted into small asolectin liposomes by dialysis against cholate-free solution. Large liposomes were produced by freezing and thawing at −80°C and room temperature, respectively. The liposomes were assayed for the SR K+ channel using the patch clamp technique. Channel density was modulated by varying protein: lipid ratios during reconstitution. Channels inserted into the membrane with a preferred orientation. The solubilised and reconstituted channel behaves ohmically over the holding potential range ±70 mV and has a conductance of 178.4±4.4 pS (mean ± SE,n=37) in 200 mM KCl. The channel has a selectivity sequence of K+>NH 4 + >Rb+>Na+ and K+ conductance is blocked by hexamethonium and decamethonium. The opening probability of the reconstituted channel is voltage dependent. The conductance and gating characteristics displayed by the solubilised and reconstituted channel correlate well with those previously observed following the fusion of native SR membrane vesicles with planar phospholipid bilayers.

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Correspondence to Barbara Tomlins.

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Tomlins, B., Williams, A.J. Solubilisation and reconstitution of the rabbit skeletal muscle sarcoplasmic reticulum K+ channel into liposomes suitable for patch clamp studies. Pflügers Arch. 407, 341–347 (1986). https://doi.org/10.1007/BF00585312

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Key words

  • Solubilised and reconstituted SR K+ channel
  • Patch clamped liposomes