Kinetic Mechanism of Na + -Glucose Cotransport through the Rabbit Intestinal SGLT1 Protein

Abstract

No consensus has yet been reached regarding the order of substrate addition to the high-affinity Na+ -D-glucose cotransporter (SGLT1). This problem was addressed by computer-assisted derivation of the steady-state velocity equations characterizing the eight-state Na+:Na+:substrate (NNS) and Na+:substrate:Na+ (NSN) mechanisms of cotransport. A notable difference was found in their denominator expressions and used to device a new strategy aimed at model discrimination in which the initial rate data are recorded at fixed S and analyzed relative to the N dependence of transport using a Hill equation. According to this protocol, the values of the Hill coefficient (n(H)) should be finite at all S (1.0 < n(H) < or =2.0) or decrease down to a limit value of 1.0 at high S in the case of the NNS and NSN models, respectively. These key experiments were performed in rabbit intestinal brush border membrane vesicles and demonstrated that a Hill equation with n(H) = 2.0 best describes the steady-state kinetics of Na+ -glucose cotransport at all S. We therefore propose a kinetic mechanism whereby Na+ binding should occur with very strong cooperativity within a rapid equilibrium segment of the transport cycle and be followed by a slow isomerization step before glucose addition.

Substrate interactions of the electroneutral Na+‐coupled inorganic phosphate cotransporter (NaPi‐IIc)

“…This conclusion is further supported by the results of the complementary experiment in which the external [Na + ] was varied at two [P i ] and no ‘ V max ’ effect was observed. This behaviour is also consistent with rapid equilibrium conditions applying to at least the P i interaction with NaPi‐IIc (for discussion, see Berteloot, 2003). As summarized in Table 1, for the P i dependence assays, the ratio of V max in ND50 to that in ND100 for the WT NaPi‐IIc, and mutant S437C were similar whereas, for the equivalent measurement using the electrogenic NaPi‐IIa, the ratio was higher (Forster et al 1998).…”