ENERGETIC COUPLING OF NA-GLUCOSE COTRANSPORT

(1) Energetic coupling in Na-linked glucose transport in renal brush border membrane vesicles has been studied in terms of various carrier models differing with respect to reaction order (random vs. ordered), and to rate limitation of steps within the routes of carrier-mediated solute transfer (translation across the membrane barrier vs. binding/release between carrier and bulk solution). (2) By computer simulation it was found that effective energetic coupling requires the leakage routes to be significantly, if not predominantly, rate-limited by their (barrier-crossing) translatory steps. This does not apply to the transfer route of the ternary complex, as coupling is possible whether or not this route is rate-limited by the translatory step. (3) The system transports glucose in the absence of Na+ (uniport) and the unidirectional flux is stimulated by unlabeled glucose on the trans side (negative tracer coupling). It is concluded that glucose binds to the carrier on either side without Na, as would be consistent with either a random system or one mode of ordered system with mirror symmetry (glucose binds before Na) but inconsistent with either mode of glide symmetry. The tracer coupling appears to indicate that the rate coefficient of carrier-mediated glucose transfer exceeds that of the empty carrier. (4) The Na-linked zero-trans flow of glucose in either direction is strongly trans-inhibited by Na. This consistent with a random system in which Na blocks or retards the translocation of the glucose-free carrier, thereby reducing 'slipping' through an internal leakage route. It is also consistent with the above mentioned ordered system, (i.e., in the absence of Na-transport without D-glucose) if it is assumed that trans Na interferes with the dissociation of the ternary complex. thereby slowing the release of glucose. (5) Minimum equilibrium exchange of glucose is stimulated in the presence of Na. This appears to indicate that Na expands the flow density of carrier-mediated glucose transfer. This expansion does not result from a 'velocity effect' (the ternary complex moving faster than the binary glucose carrier complex), as Na fails to stimulate maximum equilibrium exchange. It can instead be accounted for by an 'affinity effect' (the affinity of the carrier for glucose being increased by Na) as Na depresses the Michaelis constant of equilibrium exchange. (6) The data support the assumption that energetic coupling in a random system of Na-linked glucose transport is brought about by two kinds of effects: (a) Obstruction of the internal leakage route through the glucose-free Na-carrier complex (slipping), or (b) by expansion of the flow of the ternary Na-glucose-carrier complex by positive cooperativity. In the specified ordered system, the two effects reduce to one, as they both result from the failure of Na to bind to the glucose-free (empty) carrier. (7) Whereas the random model is consistent with all experimental observations, the ordered system appears to be inconsistent with some observations, and consistent with others only under imporbable assumptions, for instance that the final release of glucose be slow enough to limit the overall transport rate. Though a rigorous proof may still be missing, the presented evidence appears to strongly favor the random-model. (8) Kinetic data alone do not tell to which extent any of the transfer routes is rate-limited by (barrier-crossing) translatory (T) steps and by binding/release (B) steps. It can be shown theoretically, however, that effective energetic coupling requires the leakage routes, i.e., those with either glucose alone or Na alone, to be significantly, if not predominantly rate-limited by their (barrier-crossing) translatory (T) steps. It is plausible, but cannot be fully ascertained yet, that this also applies to the (transport-effective) transfer route of the ternary complex.