The Stern equation, a combination of the Langmuir adsorption isotherm, the Boltzmann relation, and the Grahame equation from the theory of the diffuse double layer, provides a simple theoretical framework for describing the adsorption of charged molecules to surfaces. The ability of this equation to describe the adsorption of divalent cations to membranes containing brain phosphatidylserine (PS) was tested in the following manner. Charge reversal measurements were first made to determine the intrinsic 1:1 association constants of the divalent cations with the anionic PS molecules: when the net charge of a PS vesicle is zero one-half of the available sites are occupied by divalent cations. The intrinsic association constant, therefore, is equal to the reciprocal of the divalent cation concentration at which the mobility of a PS vesicle reverses sign. The Stern equation with this association constant is capable of accurately describing both the zeta potential data obtained with PS vesicles at other concentrations of the divalent cations and the data obtained with vesicles formed from mixtures of PS and zwitterionic phospholipids. Independent measurements of the number of ions adsorbed to sonicated PS vesicles were made with a calcium-sensitive electrode. The results agreed with the zeta potential results obtained with multilamellar vesicles. When membranes are formed at 20~ in 0.1 M NaCI, the intrinisc 1 : 1 association constants of Ni, Co, Mn, Ba, Sr, Ca, and Mg with PS are 40, 28, 25, 20, 14, 12, respectively.

1. Purkinje myocytes were isolated from canine Purkinje strands by collagenase exposure and gentle trituration. The myocytes were studied by a switched single-micro-electrode voltage-clamp technique at 37 degrees C in Tyrode solution containing 8 mM-K+ and 2 mM-Ca2+. 2. The dose-response relation for the cardiotonic steroid dihydroouabain (DHO) was obtained by measuring the change in membrane current caused by application of concentrations of 1-100 microM. The KD obtained in fourteen experiments was 3.7 +/- 1.1 microM (mean +/- S.E. of mean). 3. We employed 100 microM-DHO (a concentration more than 25-fold greater than the KD) to estimate the resting pump current (Ip) in the isolated myocytes. A value of 0.27 +/- 0.02 microA microF-1 (mean +/- S.E. of mean, n = 32) was obtained. 4. Myocytes were also exposed to K+-free solution for a period of 200 s. On return to K+-containing Tyrode solution there was a slowly decaying outward current. The time constant of decay of this pump current transient was 87 +/- 8 s (mean +/- S.E. of mean, n = 8). The integral beneath this transient was used to obtain a second estimate of the resting pump current. In four preparations where exposures in DHO and in K+-free solutions were employed the ratio Ip, DHO/Ip, K-free was 1.76 +/- 0.15 (mean +/- S.E. of mean). 5. From the magnitude of resting pump current, in the presence of total pump blockade the Na+ activity should rise at a rate of 1.3 mM min-1. 6. Reducing [K+]o from 8 to 1 mM reduced Ip by more than 40% initially. Ip then slowly increased over the next 30 min. These results suggest that the steady-state inward background current is not greatly altered by changes in [K+]o, and that [Na+]i rises to a new level. The changes in Ip obtained at early times following reduction of [K+]o to 1 or 0.5 mM (t less than 1.75 min) were used to estimate the Km for external K+; a value of 0.8 mM was obtained. 7. The results suggest that the properties of the Na+-K+ pump in isolated canine Purkinje myocytes are similar to those in canine Purkinje strands. This argues against major distortions of measured pump properties in the canine Purkinje strand and for the physiological state of the Na+-K+ pump in the isolated Purkinje myocyte.

Recent investigations have demonstrated substantial reductions in internal [K+] in cardiac Purkinje fibers during myocardial ischemia (Dresdner, K.P., R.P. Kline, and A.L. Wit. 1987, Circ. Res. 60: 122-132). We investigated the possible role these changes in internal K+ might play in abnormal electrical activity by studying the effects of both internal and external [K+] on the gating of the inward rectifier iK1 in isolated Purkinje myocytes with the whole-cell patch-clamp technique. Increasing external [K+] had similar effects on the inward rectifier in the Purkinje myocyte as it does in other preparations: increasing peak conductance and shifting the activation curve in parallel with the potassium reversal potential. A reduction in pipette [K+] from 145 to 25 mM, however, had several dramatic previously unreported effects. It decreased the rate of activation of iK1 at a given voltage by several-fold, reversed the voltage dependence of recovery from deactivation, so that the deactivation rate decreased with depolarization, and caused a positive shift in the midpoint of the activation curve of iK1 that was severalfold smaller than the associated shift of reversal potential. These changes suggest an important role of internal K+ in gating iK1 and may contribute to changes in the electrical properties of the myocardium that occur during ischemia.

SUMMARY1. The actions of Ba2+ and Rb+, two blockers of background K+ conductance, were investigated. Recent studies performed on ungulate Purkinje fibres have suggested that the pace-maker current is an inward current activated by hyperpolarization. This hypothesis is based on the assumption that Ba2+ reduces the inwardly rectifying background K+ conductance without affecting the pace-maker current.2. Addition of 5 mM-BaCl2 to the bathing Tyrode solution decreases background K+ permeability and eliminates the reversal of the pace-maker current. The reversal reappears on return to Ba2+-free Tyrode solution. 5 mM-BaCl2 also reduces the time-dependent current at pace-maker potentials positive to about -95 mV in 4 mM-K+ Tyrode solution.3. The pace-maker current in Ba2+ Tyrode solution usually does not have an exponential time course, and often decays non-monotonically. It can take more than two minutes to reach a steady state. The fast initial component of membrane current, which is observed on hyperpolarizing in the pace-maker potential range in Purkinje fibres and which has been called the 'depletion current', is still present in Ba2+ Tyrode solution, but is reduced or eliminated if 10 mM-CsCl is added to the Ba2+ Tyrode solution. The addition of Cs+ is accompanied by an outward shift in membrane current in Ba2+ Tyrode solution.4. Ba2+ reduces the background K+ permeability in a dose-dependent manner. Addition of between 0-5 and 1 mM-BaCl2 achieves a maximum effect. Raising the amount of BaCl2 above this level reduces the time-dependent current even when no further effect on background permeability is observed.5. Rb+ substitution for K+ reduces the magnitude of the pace-maker current at potentials positive to -100 mV, eliminates the reversal of the pace-maker current, shifts the activation range to more negative potentials, and decreases the voltage dependence of pace-maker current kinetics. Rb+ addition to Tyrode solution has little effect on pace-maker current magnitude or time course positive to -90 mV, but does shift the reversal to more negative potentials.6. The available evidence suggests that the pace-maker current in Ba2+ Tyrode solution is an inward current activated by hyperpolarization. However, Ba2+ blocks an unknown fraction of the pace-maker current in a dose-dqpendent, and possibly voltage-dependent manner. Also, the presence of a slow component of pace-maker