Characterization of a high-conductance,voltage-dependent cation channel from the plasma membrane of rye roots in planar lipid bilayers

Plasma-membrane vesicles were purified by aqueous-polymer two-phase partitioning of a microsomal membrane fraction from rye (Secale cereale L.) roots and incorporated into planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers. A high-conductance cation channel (a maxi cation channel) was characterized from single-channel electrical recordings. The channel was incorporated into the bilayer with its cytoplasmic surface facing the trans compartment and voltages were referenced cis with respect to trans. The channel was permeable to both monovalent and divalent cations. The unitary conductance was 451 pS in symmetrical 100 mM KCl and 213 pS in symmetrical 100 mM BaCl₂. The permeability ratio P_KP_Ba was 1.002.56. Unitary conductances declined in the order K⁺Rb⁺>Cs⁺>Na⁺> Li⁺ (monovalent cations) and Ba²⁺>Sr²⁺>Ca²⁺> Mg²⁺>Co²⁺>Mn²⁺ (divalent cations). The relative permeabilities of monovalent cations mirrored their conductivity sequence, whereas the permeabilities of all divalent cations were similar. The maxi cation channel showed complex kinetics, exhibiting both voltage- and time-dependent inactivation and voltage-dependent gating. The voltage dependence of the kinetics shifted in parallel with changes in the reversal potential of the channel. In symmetrical 100 mM KCl, following a voltage step from zero to the test voltage, the channel inactivated and the active-channel lifetime ( _i) shortened exponentially as the test voltage was increased. The channel always opened immediately upon depolarization to zero volts, indicating that inactivation of the channel did not result from the loss of any intrinsic factor. The probability of finding an active channel in the open state (P₀) exhibited a bell-shaped relationship with membrane potential. At voltages between -40 and 80 mV, P₀ exceeded 0.99, but p₀ declined abruptly at more extreme voltages. Under ionic conditions which approximated physiological conditions, in the presence of 100 mM KCl on the trans (cytoplasmic) side and 1 mM KCl plus 2 mM CaCl₂ on the cis (extracellular) side, the reversal potential was 15.6 mV and the kinetics approximated those observed in symmetrical 100 mM KCl. Thus, the channel would open upon depolarization of the plasma membrane in vivo. If the channel functioned physiologically as a Ca²⁺ channel it might be involved in intracellular signalling: the channel could open in response to a variety of environmental, developmental and pathological stimuli which depolarize the plasma membrane, allowing Ca²⁺ into the cytoplasm and thereby initiating a physiological response.Abbreviations E_K Nernst (equilibrium) potential for potassium - E_rev zero-current (reversal) potential - I/V current/voltage - _c apparent mean lifetime of the activated-channel closed state - _i apparent mean lifetime of the activated channel following a voltage step from zero volts - ₀ apparent mean lifetime of the activated-channel open state - PE 1-palmitoyl-2-oleoyl phosphatidylethonlamine - P₀ probability of finding the activated channel in an open state - TEA⁺ tetraethylammonium This work was supported by the Agriculture and Food Research Council and by a grant from the Science and Engineering Research Council Membrane Initiative (GR/F 33971) to Prof. E.A.C. MacRobbie (University of Cambridge, UK).