In Xenopus oocytes, stimulation of the electrogenic Na/HCO3 transporter NBCe1 by IRBIT can be explained by relief of transporter autoinhibition and is unaffected by endogenous phosphatases.

Abstract

Other have shown in Xenopus oocytes that NBCe1 lacking an amino‐terminal (Nt) autoinhibitory sequence has a ~3‐fold greater transport activity than full‐length NBCe1. In a separate study, it was reported that co‐expression of full‐length NBCe1 with its Nt binding partner IRBIT results in a ~7‐fold stimulation of transporter activity. At face value these data suggest that the stimulatory action of IRBIT cannot be explained solely by relief of autoinhibition. However, these data pertain to different splice variants of NBCe1.In the present study, we work only with the pancreatic splice form NBCe1‐B. We find that, compared to oocytes expressing full‐length NBCe1‐B alone, oocytes expressing (1) a mutant NBCe1‐B that lacks the autoinhibitory sequence manifest a 7.5‐fold greater transporter activity (2) full‐length NBCe1‐B plus IRBIT manifest a 5.2‐fold greater transporter activity.Oocytes have endogenous protein phosphatase 1 (PP1) and PP1 is reported to deactivate IRBIT following binding at a non‐consensus PP1‐binding site ‘KQIQF’ in IRBIT. Thus we hypothesized that the full stimulatory effect of IRBIT was masked by the action of endogenous PP1. However, we find that removing the putative PP1 binding site in IRBIT does not result in a substantial increase in NBCe1‐B stimulation. Conversely an IRBIT in which we replaced ‘KQIQF’ with a well characterized PP1‐binding consensus sequence of the type ‘RVxF’ was unable to stimulate NBCe1‐B activity.In conclusion, our findings are consistent with a model in which the action of IRBIT is explained solely by relief of autoinhibition. The results are not confounded by endogenous oocyte PP1 activity, because the endogenous PP1 has no detectable functional interaction with the ‘KQIQF’ motif in overexpressed IRBIT.