Graded amplification of the Na, K‐ATPase across a subclonal series: Effects on membrane physiology

Abstract

We have generated a series of clonally related cell lines which differ in the level of amplified expression of the Na,K-ATPase. These lines, originally derived from the ouabain resistant HeLa variant C+, expressed different numbers of binding sites for the Na,K-ATPase inhibitor ouabain, ranging from 2.9 X 10(6)/cell to 11.8 X 10(6)/cell. Amplification of the genes for both subunits of the enzyme was also seen but was not strictly correlated with level of expression. The influxes of histidine and tetraphenylphosphonium were measured across a series, including HeLa S3 and revertants, expressing from 0.74 X 10(6) to 10.5 X 10(6) ouabain-binding sites per cell. Tetraphenylphosphonium influx rate, presumed to be a function of membrane potential, varied linearly with ouabain binding site number, while histidine influx varied with the log of ouabain binding site number. Our results suggest that membrane potential increases in a simple fashion across our series of amplified lines. However, histidine influx was unaffected by treatments which cause membrane depolarization and a decrease in tetraphenylphosphonium influx rate. We propose that increasing histidine influx rates across our amplified series reflects exchange acceleration of L system transport due to increased intracellular pools of L system reactive amino acids. The Na,K-ATPase is ultimately responsible for most active transport across the plasma membrane. The consistent, graded physiological alterations seen across this series of closely related lines, chosen for graded enzyme expression, demonstrate the value of this novel genetic approach to the study of the energization of membrane transport.

Steady-state physiological variations across a graded series of Na,K-ATPase-amplified cells.

“…Histidine is transported only by system L in these lines, and histidine flux rate is a function of the number of ouabain-binding sites (20). We had proposed that histidine flux was a direct function of the pool sizes of system (Fig.…”

“…All lines used in this study were derived from the human carcinoma line HeLa S3 (23) Isolation of subclonal lines. Subclonal stably ouabainresistant lines were isolated as previously described (20). Amplified lines used in these studies were all subclones of Cl (21).…”

“…Previously, we have shown that the influx rate of histidine is a function of the number of ouabain-binding sites in a subclonal series (20). Since histidine transport in HeLa cells is sodium independent, this result might seem surprising.…”

“…Subsequently, it was shown that resistance to ouabain in these lines is based on amplified expression (21) of the sodium-and potassiumactivated ATPase (Na,K-ATPase), correlated with amplification of the genes for both the alpha (8,20,21,28) and beta (19,20) subunits of the enzyme. The cardiac glycoside resistance based on amplification of the Na,K-ATPase genes is clearly different in nature from that produced by singlestep selection of mutagenized HeLa cells (11,26).…”

“…Recently, we described relationships between some physiological functions of the plasma membrane and ouabainbinding site number (a measure of expression of the Na,KATPase) in a series of subclones which express graded levels of the Na,K-ATPase (20). The NA,K-ATPase is responsible for generating and maintaining the gradients of sodium and potassium and the membrane potential across the plasma membrane.…”