ALBERT

Notes: Summary Modulation of the current generated by the Na+/K+ pump by membrane potential and protein kinases was investigated in oocytes of Xenopus laevis. In addition to a positive slope region in the current-voltage (I–V) relationship of the Na+/K+ pump, a negative slope region has been described in these cells (Lafaire & Schwarz, 1986) and has been attributed to a voltage-dependent apparent K m value for pump stimulation by external [K+] (Rakowski et al., 1991). To study this feature in more detail. Xenopus oocytes were used for comparative analysis of the negative slope of the I–V relationship of the endogenous Na+/K+ pump and of the Na+/K+ pump of the electric organ of Torpedo californica expressed in the oocytes. The effects of stimulation of protein kinases A and C on the negative slope were also analyzed. To investigate the negative slope over a wide potential range, experiments were performed in Na+-free solution and in the presence of high concentrations of Ba2+ and tetraethylammonium, to block all nonpump related K+-sensitive currents. Pump currents and pump-mediated fluxes were determined as differences of currents or fluxes in solutions with and without extracellular K+. The voltage dependence of the K m value for stimulation of the Na+/K+ pump by external [K+] shows significant species differences. Over the entire voltage range from -140 to +20 mV, the K m value for the Na+/K+ pump of Torpedo electroplax is substantially higher than for the endogenous pump and exhibits more pronounced voltage dependence. For the Xenopus pump, the voltage dependence can be described by voltage-dependent stimulation by external [K+] and can be interpreted by voltage-dependent K+ binding, assuming that an effective charge between 0.37 and 0.56 of an elementary charge is moved in the electrical field. An analogous evaluation of the voltage dependence of the Torpedo pump requires the assumption of movement of two effective charges of 0.16 and 1.0 of an elementary charge. Application of 1,2-dioctanoyl-sn-glycerol (diC8. 10–50 μm). which is known to stimulate protein kinase C, reduces the maximum activity of the Xenopus pumps in the oocyte membrane by 40% and modulates the voltage dependence of K+ stimulation. For the endogenous Xenopus pump, the apparent effective charge increased from 0.37 to 0.51 of elementary charge and the apparent K m at 0 mV increased from 0.46 to 0.83 mm. For the Torpedo pump, one of the apparent effective charges increased from 1.0 to 2.5 of elementary charge. Injection of cAMP (final concentration 50 μm), which stimulates protein kinase A, has an effect opposite to stimulation of protein kinase C. The activity of the Xenopus Na+/K+ pump is elevated by 80%, and the voltage dependence of K+ stimulation becomes less pronounced. For the endogenous pump the apparent effective charge decreased from 0.56 to 0.38 of elementary charge and the apparent K m at 0 mV decreased from 0.78 to 0.65 mm. Also for the Torpedo pump, the effective charges and apparent affinities became reduced. The data suggest that species differences in voltage-dependent stimulation of the Na+/K+ pump by external K+ can account for differences in the steepness of the negative slope in the I–V relationships observed in different preparations. In addition, they suggest that the voltage dependence and the maximum activity of the Na+/K+ pump can be modulated by activation of protein kinases.

Notes: Abstract Exact predictions for two-pulse visual temporal integration data are derived from the Bouman-van der Velden quantum coincidence model for threshold vision. The predictions of the model start with complete summation for superposed pulses, then pass to a transition zone of partial integration, and finally reach the level of probability summation for pulses presented with large interstimulus intervals. From our results we can clearly reject the assumption of constant integration times with the basic model. We thus generalize the coincidence model to allow for variable integration times, derive the corresponding predictions for two-pulse integration data, and compare these predictions to published data currently available. It is shown that detectors of low order of coincidence generally underestimate the actual reduction of threshold intensity (or equivalently the corresponding increase of the detection probability) for two pulses as compared to the singlepulse performance.

Notes: Abstract Currents generated by the Na+/K+ ATPase were measured under voltage clamp in oocytes of Xenopus laevis. The dependence of pump current on external [Na+] was investigated for the endogenous Xenopus pump as well as for wild-type and mutated pumps of electroplax of Torpedo californica expressed in the oocytes. The mutants had α-subunits truncated before position Lys28 (αΔK28) or Thr29 (αΔT29) of the N-terminus. The currents generated by all variants of pump molecules in the presence of 5 mM K+ show voltage-dependent inhibition by external [Na+]. The apparent K1 values increase with membrane depolarisation, and the potential dependence can be described by the movement of effective charges in the electrical potential gradient across the membrane. Taking into account Na+-K+ competition for external binding to the E2P form, apparent K1 values and effective charges for the interaction of the Na+ ions with the E2P form can be estimated. For the Xenopus pump the effective charge amounts to 1.1 of an elementary charge and the K1 value at 0 mV to 44 mM. For the wild-type Torpedo pump, the analysis yields values of 0.73 of an elementary charge and 133 mM, respectively. Truncation at the N-terminus removing a lysinerich cluster of the a-subunit of the Torpedo pump leads to an increase of the effective charge and decrease of the K1 value. For αΔK28, values of 0.83 of an elementary charge and 117 mM are obtained, respectively. If LyS28 is included in the truncation (α·T29), the effective charge increases to 1.5 of an elementary charge and the apparent K1 value is reduced to 107 mM. The K, values for pump inhibition by external Na+, calculated by taking into account Na+-K+ competition, are smaller than the K/12 values determined in the presence of 5 mM [K+]. The difference is more pronounced for those pump variants that have higher Km, values. The variations of the parameters describing inhibition by external [Na+] are qualitatively similar to those described for the stimulation of the pumps by external [K+] in the absence of extracellular [Na+]. The observations may be explained by an acess channel within the membrane dielectric that has to be passed by the external Na+ and K+ ions to reach or leave their binding sites. The potential-dependent access and/or the interaction with the binding sites shows species differences and is affected by cytoplasmic lysine residues in the N-terminus.