Permeability of reconstituted sarcoplasmic reticulum vesicles: Reconstitution of the K +, Na + channel

Abstract

Permeability properties of reconstituted rabbit skeletal muscle sarcoplasmic reticulum vesicles were characterized by measuring efflux rates of [ 3H]inulin, [ 3H]choline +, 86Rb +, and 22Na +, as well as membrane potential changes using the voltage-sensitive probe, 3,3′-dipentyl-2,2′-oxacarbocyanine. Native vesicles were dissociated with deoxycholate and were reconstituted by dialysis. Energized Ca 2+ accumulation was partially restored. About 1 2 of the reconstituted vesicles were found to be ‘leaky’, i.e., permeable to choline + or Tris + but not to inulin. The remaining reconstituted vesicles were ‘sealed’, i.e., impermeable to choline +, Tris + and inulin. Sealed reconstituted vesicles could be further subdivided according to their K +, Na + permeability. About 1 2 , previously designated Type I, were readily permeable to K + and Na +, indicating the presence of the K +, Na + channel of sarcoplasmic reticulum. The remaining sealed vesicles (Type II) formed a permeability barrier to K + and Na +, suggesting that they lacked the K +, Na + channel. These studies show that the K +, Na + channel of sarcoplasmic reticulum can be solubilized with detergent and reconstituted with retention of activity. Furthermore, our results suggest that part or all of the decreased Ca 2+-loading efficiency of reconstituted vesicles may be due to the presence of a significant fraction of leaky vesicles.