Temperature effects on the kinetic properties of the rabbit intestinal oligopeptide cotransporter PepT1

Abstract

The effects of temperature on the functional properties of the intestinal oligopeptide transporter PepT1 from rabbit have been investigated using electrophysiological methods. The dipeptide Gly-Gln at pH 6.5 or 7.5 was used as substrate. Raising the temperature in the range 20-30 °C causes an increase in the maximal transport-associated current (I (max)) with a Q (10) close to 4. Higher temperatures accelerate the rate of decline of the presteady-state currents observed in the absence of organic substrate. The voltage dependencies of the intramembrane charge movement and of the time constant of decline are both shifted towards more negative potentials by higher temperatures. The shift is due to a stronger action of temperature on the outward rate of charge movement compared to the inward rate, indicating a lower activation energy for the latter process. Consistently, the activation energy for the complete cycle is similar to that of the inward rate of charge movement. Temperature also affects the binding rate of the substrate: the K (0.5) -V curve is shifted to more negative potentials by higher temperatures, resulting in a lower apparent affinity in the physiological range of potentials. The overall efficiency of transport, estimated as the I (max)/K (0.5) ratio is significantly increased at body temperature.

An Inverse Relationship Links Temperature and Substrate Apparent Affinity in the Ion-Coupled Cotransporters rGAT1 and KAAT1

“…The results illustrated above for rGAT1 and KAAT1 confirm the effect already observed in rabbit PepT1 [12] and suggest that a change in apparent affinity with temperature might be a feature shared by different transporters, even those belonging to diverse gene families. It must be noted, however, that some results indicating no significant effects have been also reported in other transporters [25].…”

“…As illustrated in Figure 2C, raising the temperature from 20 to 30 °C produces a considerable increase in I max , which is, however, not as large as in the case of rGAT1 (Figure 1C), but is, instead, similar to that observed in the oligopeptide transporter PepT1 [12], with Q 10 values between 3 and 4. The action of temperature on K 05 is shown in Figure 2D.…”

“…For these experiments, a staircase voltage protocol was used to minimize temperature fluctuations during the recording [12]. For rGAT1, the protocol was applied from a holding potential ( V h ) = −40 mV and consisted of five steps spanning the range from −120 to +40 mV in 40 mV intervals.…”

“…, the electrical signal arising from the initial steps of the transport cycle [1,10,11]. In a recent work from our laboratory on the rabbit intestinal oligopeptide transporter PepT1 [12], we observed that the apparent substrate affinity was significantly affected by temperature. In particular, a decrease in apparent affinity was measured in the physiological voltage range when increasing the temperature from 20 to 30 °C.…”

Conferring electrogenicity to the electroneutral phosphate cotransporter NaPi-IIc (SLC34A3) reveals an internal cation release step

“…Solute coupled carrier proteins generally exhibit a high temperature dependence that differs significantly from that of ion channels and can be used to distinguish between diffusional processes and those involving conformational changes (3,5,6,21). With respect to steady-state cotransport rate (I P i ), we estimated that E a for the electrogenic human NaPi-IIa and AAD-IIc was respectively 2-and 3-fold larger than for the corresponding P i uptake rate of the electroneutral NaPi-IIc (Fig 2C).…”