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

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 Ca²⁺ accumulation was partially restored. About $\text{1}\text{2}$ 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 $\text{1}\text{2}$, 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 Ca²⁺-loading efficiency of reconstituted vesicles may be due to the presence of a significant fraction of leaky vesicles.