Enzyme kinetics of the prime K+ channel in the tonoplast of Chara: Selectivity and inhibition

Summary The prime potassium channel from the tonoplast of Chara corallina has been analyzed in terms of an enzyme kinetic model (Gradmann, Klieber & Hansen 1987, Biophys. J. 53:287) with respect to its selectivity for K⁺ over Rb⁺ and to its blockage by Cs⁺ and by Ca²⁺. The channel was investigated by patchclamp techniques over a range of membrane voltages (V _m , referred to an extracytoplasmic electrical potential of zero) from –200 mV to + 200 mV under various ionic conditions (0 to 300 mM K⁺, Rb⁺, Cs⁺, Ca²⁺, and Cl^–) on the two sides of isolated patches. The experimental data are apparent steady-state currentvoltage relationships under all experimental conditions used and amplitude histograms of the seemingly noisy open-channel currents in the presence of Cs⁺. The used model for K⁺ uniport comprises a reaction cycle of one binding site through four states, i.e., (1) K⁺-loaded and charged, facing the cytoplasm, (2) K⁺-loaded and charged facing the vacuole, (3) empty, facing the vacuole, and (4) empty, facing the cytoplasm. V _m enters the system in the form of a symmetric Eyring barrier between state 1 and 2. The numerical results for the individual rate constants are (in 10⁶s^–1 for zero voltage and 1 m substrate concentration): k ₁₂: 1,410, k ₂₁: 3,370, k ₂₃: 105,000, k ₃₂: 10,600, k ₃₄: 194, k ₄₃: 270, k ₄₁: 5,290, k ₁₄: 15,800. For the additional presence of an alternate transportee (here Rb⁺), the model can be extended in an analog way by another two states ((5) Rb⁺-loaded and charged, facing cytoplasm, and (6) Rb⁺-loaded and charged, facing vacuole) and six more rate constants (k ₄₅: 300, k ₅₄: 240, k ₅₆: 498, k ₆₅: 4,510, k ₆₃: 4,070, k ₃₆: 403). This six-state model with its unique set of fourteen parameters satisfies the complete set of experimental data. If the competing substrate can be bound but not translocated (here Cs⁺ and Ca²⁺), k ₅₆ and k ₆₅ of the model are zero, and the stability constants K _cyt (= k ₃₆/k ₆₃) and K _vac (= k ₄₅/k ₅₄) turn out to be K _cyt(Ca²⁺): 250 m ^–1 · exp(V _m /(64 mV)), k _vac(Ca²⁺): 10 m ^–1 · exp(–V _m /(66 mV)), K _cyt(Cs⁺): 0, and K _vac(Cs⁺): 46 m ^–2 · exp(–V _m /(12.25 mV)). With the assumption that the current fluctuations in the presence of Cs⁺ consist of incompletely resolved, short periods of complete openings and complete closures, the amplitude histograms of the noisy open channel currents can be described by a beta distribution, yielding the rate constants for binding (92 · 10⁶ sec^–1 · m ^–2 · exp(–V _m /(22.5 mV)) and debinding (2, 10⁶ sec^–1 · m ^–2 · exp(V _m /(22.5 mV)) of Cs⁺ to the vacuolar side of the channel as functions of the Cs⁺] and of V _m . Considering these data and those from the literature, an asymmetry of the channel can be assessed, with a high charge density at the cytoplasmic side (Eisenman-series Nr. XI) and a low charge density at the vacuolar side (Eisenman-series Nr. I). Furthermore, the results provide an example for intimate linkage between conduction and switching of a channel.This work has been supported by the Deutsche Forschungsgemeinschaft.