If both protonation steps were subject to the diffusive barrier, the best-fitting distance would be smaller. == Additional information == == Competing interests == None declared. == Author contributions == All the experiments were carried out in the Department of Physiology, Anatomy and Genetics, University of Oxford. with a model where CAicatalyses local H+iondeliveryto the NBC protein, assisting the subsequent (uncatalysed) protonation and removal of imported HCO3ions. In well-superfused myocytes, exofacial CA activity is superfluous, most likely because extracellular CO2/HCO3buffer is clamped at equilibrium. The CAiinsensitivity of NHE flux suggests that, in the native cell, intrinsic mobile buffer-shuttles supply sufficient intracellular H+ions to this transporter, while intrinsic buffer access to NBC proteins is restricted. Our results demonstrate a selective CA facilitation of acid/base transporters in the ventricular myocyte, implying a specific role for the intracellular enzyme in HCO3transport, and hence pHiregulation in the heart. == Introduction == Carbonic anhydrase (CA) proteins are expression products of a Indeglitazar gene-family that codes for at least 16 isoforms (Sly & Hu,1995; Supuran,2008). All except CAVII, CAX and CAXI are functionally active enzymes, widely expressed, which catalyse the reversible hydration of CO2to H+and HCO3ions. Catalytically active CAs fulfil a number of functions (Maren,1967). They can accelerate the cellular venting of CO2(Geers & Gros,2000; Swietachet al.2008,2009), the spatial diffusion of intracellular and extracellular H+ions (Stewartet al.1999; Spitzeret al.2002; Swietachet al.2009; Hulikovaet al.2011), and the speed of CO2/HCO3(carbonic) buffering (Leem & Vaughan-Jones,1998). A major role for CAs is proposed to be the catalytic enhancement of acid/base membrane transport. Some reports suggest they achieve this by binding directly to pH regulatory transporters, forming a multimeric protein complex Indeglitazar called a transport metabolon (Sterlinget al.2001,2002; Morganet al.2007; Caseyet al.2009; Svastovaet al.2012; Vargas & Alvarez,2012). The proposal remains controversial, as observations of direct CA binding and flux facilitation have recently been challenged (Boron,2010; Al-Samiret al.2013; Parker & Boron,2013), while the molecular mechanisms involved are far from clear. In some cases, for example, CA protein may stimulate acid transport allosterically, even in the absence of enzymic activity (Beckeret al.2011). Various CA isoforms are expressed in mammalian heart. In ventricular myocytes, CAII is a cytosolic soluble intracellular isoform, CAIV and XIV are sarcolemmally tethered with exofacially exposed catalytic sites, while CAXIV and IX have also been detected at sarcoplasmic reticular and possibly transverse tubule membranes (Scheibeet al.2006; Schroederet al.2013). CA activity in these cells has been shown to accelerate regulatory pHirecovery following intracellular acid or base loads (Lagadic-Gossmannet al.1992; Orlowskiet al.2011; Vargas & Alvarez,2012). Moreover, the principal acid-extruding transporters in these cells, Na+/H+exchange (NHE1 isoform) and Na+HCO3cotransport (NBCe1 and NBCn1) (Aielloet al.1998; Yamamotoet al.2005; De Giustiet al.2011; Garciarenaet al.2013; for review see Vaughan-Joneset al.2009), when co-expressed heterologously with intracellular CAII or exofacial CAIV Sav1 in oocytes or cultured cell lines, exhibit enhanced activity that is slowed in the presence of pharmacological CA inhibitors such as acetazolamide (ATZ) (Grosset al.2002; Liet al.2002; Alvarezet al.2003; Loiselleet al.2004; Liet al.2006; Becker & Deitmer,2007; Schueleret al.2011). It has yet to be shown, however, that NHE and NBC activities are specifically regulated by either intracellular or exofacial CA in native ventricular cells. In the present work we have examined the influence of the catalytic activity of intracellular and exofacial CA (CAiand CAe, respectively) on NHE-and NBC-mediated acid efflux from rat ventricular myocytes. Intrinsic transporter and CA activity in these cells has been assessed from measurements of intracellular pH, intracellular [Na+], and extracellular surface pH (pHe) using Indeglitazar ion-selective fluorophores (Leemet al.1999; Yamamotoet al.2005; Schroederet al.2013), while applying membrane-permeant and impermeant CA inhibitors to gauge the relative importance of CAiand CAe. Results suggest a novel and selective role for intracellular catalytic CA activity in controlling membrane acid extrusion, while questioning any obligatory role.