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

Abstract

The prime potassium channel from the tonoplast of Chara corallina has been analyzed in terms of an enzymatic 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 patch-clamp techniques over a range of membrane voltages (Vm, 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 current-voltage 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. Vm 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 10(6)s-1 for zero voltage and 1 M substrate concentration): k12: 1,410, k21: 3,370, k23: 105,000, k32: 10,600, k34: 194, k43: 270, k41: 5,290, k14: 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 (k45: 300, k54: 240, k56: 498, k65: 4,510, k63: 4,070, k36: 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+). k56 and k65 of the model are zero, and the stability constants Kcyt (= k36/k63) and Kvac (= k45/k54) turn out to be Kcyt(Ca2+): 250 M-1 x exp(Vm/(64 mV)), kvac(Ca2+): 10 M-1 x exp(-Vm/(66 mV)), Kcyt(Cs+): 0, and Kvac(Cs+): 46 M-2 x exp(-Vm/(12.25 mV)).(ABSTRACT TRUNCATED AT 400 WORDS)