Properties of the Ca²⁺-activated K⁺ channel in one-step inside-out vesicles from human red cell membranes

Abstract

The presence of a calcium-activated potassium channel in some mammalian red cell membranes makes them useful experimental models in which to study the properties of similar mechanisms believed to be involved in the control of membrane potential and conductance, at rest and during activity, in many other cells. However, vesicles prepared from human red cell membranes by the method of Steck et al.¹, whether inside-out (IOVs) or right-side out (ROVs), either failed to show any Ca²⁺-activated component of K⁺(Rb⁺) fluxes² or showed only a very reduced calcium sensitivity and K⁺/Na⁺ selectivity³. This is surprising because there is good functional preservation of other transport mechanisms, such as the anion carrier⁴, Ca²⁺ pump⁵ and Na⁺ pump⁶. Our failure to confirm reports⁷ that the calcium response could be restored in ‘silent’ IOVs by addition of protein concentrates from red cell lysates prompted the search for and discovery of vesiculation procedures which produced ion-tight IOVs in a single step, with minimum loss of membrane components and transport properties^8–11. We report here (1) the conditions which favour preservation or loss of the Ca²⁺-activated component of the ⁸⁶Rb efflux from one-step IOVs, (2) an approximate estimate of the number of Ca²⁺-activated K⁺ channels per red cell, and (3) that individual Ca²⁺-activated K⁺ channels respond in an all or nothing fashion to Ca²⁺ activation and differ in their threshold sensitivity to ionized calcium.