Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: I. Tight-seal whole-cell recordings

Abstract

Electrical currents associated with sodium-coupled alanine transport in mouse pancreatic acinar cells were studied using the method of whole-cell recording with patch pipettes. Single cells or small clusters of (electrically coupled) cells were isolated by collagenase treatment. The composition of the intracellular solution could be controlled by internal perfusion of the patch pipette. In this way both inward and outward currents could be measured under "zero-trans" conditions, i.e., with finite concentrations of sodium and L-alanine on one side and zero concentrations on the other. Inward and outward currents for equal but opposite concentration gradients were found to be of similar magnitude, meaning that the cotransporter is functionally nearly symmetric. The dependence of current on the concentrations of sodium and L-alanine exhibited a Michaelis-Menten behavior. From the sodium-concentration dependence of current as well as from the reversal potential of the current in the presence of an alanine-concentration gradient, a sodium/alanine stoichiometric ratio of 1:1 can be inferred. The finding that N-methylated amino acids may substitute for L-alanine, as well as the observed pH dependence of currents indicate that the pancreatic alanine transport system is similar to (or identical with) the "A-system" which is widespread in animal cells. The transport system is tightly coupled with respect to Na+; alanine-coupled inward flow of Na+ is at least 30 times higher than uncoupled Na+ flow mediated by the cotransporter. The current-voltage characteristic of the cotransporter could be (approximately) determined from the difference of transmembrane current in the presence and in the absence of L-alanine. The sodium-concentration dependence of the current-voltage characteristic indicates that a Na+ ion approaching the binding site from the extracellular medium has to cross part of the transmembrane electric field.

Highly Conserved Asparagine 82 Controls the Interaction of Na+ with the Sodium-coupled Neutral Amino Acid Transporter SNAT2

“…Transport of neutral amino acids by SNAT2 is coupled to the cotransport of Na ϩ ions, with a proposed coupling stoichiometry of 1 neutral amino acid to 1 Na ϩ ion (14,18,19). Although the amino acid is a neutral zwitterion, the Na ϩ ion carries charge.…”

Stimulus‐secretion coupling: cytoplasmic calcium signals and the control of ion channels in exocrine acinar cells.

“…This type of experiment was useful in excluding the existence of a special diffusional barrier for ACh surrounding the acinus since the L-alanine-evoked depolarization is less steep than that evoked by ACh in spite of the shorter latency (Iwatsuki & Petersen, 1980). The L-alanine-evoked depolarization is due to Na+-L-alanine co-transport (Iwatsuki & Petersen, 1980;Jauch, Petersen & Lauger, 1986;Boyd & Yudilevich, 1987) and this operates as soon as both Na+ and L-alanine are available. The long latency for the ACh-evoked depolarization is not due to a possible intracellular site of action since Iwatsuki & Petersen (1977 a) showed that ACh only exerts an effect on the membrane potential when added to the extracellular side of the membrane and not when injected into the cell.…”

Differential potentiation of GABAA receptor function by two stereoisomers of diimidazoquinazoline analogues

“…This is consistent with a tight electrical coupling between the cells. Otherwise, more than one exponential function is needed to describe the current decline (Jauch et al, 1986). The voltage change under current clamp conditions in a cluster of six cells ( Figure 2b) was uniform and further supports the existence of one electrical compartment in the cluster.…”