Properties of an electrogenic sodium‐potassium pump in isolated canine Purkinje myocytes.

Abstract

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.

Amino acid substitutions in the rat Na+, K(+)‐ATPase alpha 2‐subunit alter the cation regulation of pump current expressed in HeLa cells.

“…We probed the role of E327 and D925 in cation transport by substituting the charged side-chain with neutral groups, expressing these substituted enzymes (Bahinski, Nakao & Gadsby, 1988;Gasby & Nakao, 1989;Yamamoto et al 1996) (Cohen, Datyner, Gintant, Mulrine & Pennefather, 1987;Kinard et al 1994). The Kd values for both E327Q and D925L were higher, namely 7-7 and 6-4 mm, respectively.…”

Dynamic Ca 2+ -Induced Inward Rectification of K + Current During the Ventricular Action Potential

“…In spite of such complexities, on average, the effect of low extracellular Ca 2ϩ was consistent with that of Ca 2ϩ channel blockade. In the presence of low Ca 2ϩ (nϭ7, Figure 7d) It can be anticipated that besides reducing K ϩ channel conductance, removal of K ϩ o will also inhibit the Na ϩ -K ϩ pump, 18 thus resulting in a reduction of I NaK and in a progressive dissipation of the Na ϩ transmembrane gradient. A decrease in the Na ϩ gradient, in turn, will reduce the inward component of the current (I NaCa ) generated by the Na ϩ -Ca 2ϩ exchanger (ie, it will move the reversal potential of I NaCa in the negative direction).…”

Interaction of intracellular ion buffering with transmembrane-coupled ion transport.

“…Maximum suppression of Na § influx is indicated by arrows on all curves. changes in [Na]i on Na + influx than the ohmic alternative (Cohen et al, 1987a). Thus the constant field approximation should give results between those of a constant leak and an ohmic leak.…”

“…A second measure of resting pump current (Ip[0K]) can be obtained by exposing a prep-aration to K+-free Tyrode for a time, T, and then returning it to normal Tyrode. The integral (Q) of the additional pump current is then related to the resting pump current (Cohen et al, 1987a) by:…”