Abstract
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 voltage-dependent cation-channel became incorporated into the bilayer with its cytoplasmic surface facing the trans compartment (which was grounded) and was characterized from single-channel recordings. The channel had a unitary conductance of 174 pS in symmetrical 100 mM KCl. The selectivity towards monovalent cations, determined from both conductance measurements in symmetrical 100 mM cation chloride and from permeability ratios in the presence of (cis:trans) 100 mM cation chloride:100 mM KCl, was Cs ≥ K ≥ Rb > Na. The channel was also permeable to both Ba2+ and Ca2+. Although the unitary conductances in symmetrical 100 mM BaCl2 and CaCl2 were only 46 pS and 40 pS, respectively, the apparent permeabilities of the divalent cations relative to K+ were greater than expected (PK:PBa:PCa, 1.00:1.66:2.60). This anomaly might result from competition between divalent and monovalent cations for an intrapore binding site. The channel exhibited complex gating kinetics, which were modulated in response to changes in the zero-current (reversal) potential of the channel (Erev). In symmetrical 100 mM KCl the channel inactivated at positive voltages greater than 100 mV and the activated channel exhibited a high probability of being in an open-state (P0 > 0.90) at all voltages between ± 100 mV. Channel P0 approximated unity at voltages in the range - 60 to + 20 mV. As more-negative voltages were applied, P0 decreased gradually. In contrast, as more positive voltages were applied, P0 decreased initially to a local minimum (approaching P0 = 0.90), then increased as the voltage was further increased before declining at extreme positive voltages. Under physiologically relevant ionic conditions, with 100 mM KCl plus contaminant Ca2+ on the trans (cytoplasmic) side and 1 mM KCl plus 2 mM CaCl2 on the cis (extracellular) side of the channel, Erev was 25.2 mV and the relative permeability PCa/PK was 7.45. Thus, the channel would be activated by plasma-membrane depolarization in vivo and facilitate Ca2+ influx and net K+ efflux. A role in intracellular signalling is proposed for this channel. It could open in response to stimuli which depolarize the plasma membrane, allowing Ca2+ into the cytoplasm and, thereby, initiating a cellular response. The outward K+ current would act to stabilize the trans-plasma membrane voltage, preventing excessive depolarization during Ca2+ influx.