Expression and permeation properties of the K+ channel Kir7.1 in the retinal pigment epithelium

The retinal pigment epithelium (RPE) is a simple cuboidal epithelium in the distal retina that separates the photoreceptor cells from their main blood supply in the choroid. From this strategic position, the RPE carries out a host of functions that are critical to the visual process. One of these is the transepithelial transport of fluid, ions and metabolites, which serves to control the composition and volume of the extracellular fluid that surrounds the photoreceptor outer segments (Hughes et al. 1998). It is well established that K+ channels play a central role in the vectorial transport of K+ across the RPE. At the apical membrane, the net flux of K+ into or out of the subretinal space is determined by the balance between K+ efflux through Ba2+-sensitive K+ channels (Lasansky & De Fisch, 1966; Miller & Steinberg, 1977; Griff et al. 1985; Joseph & Miller, 1991; Quinn & Miller, 1992) and K+ influx via the electrogenic Na+-K+ pump (Miller et al. 1978) and Na+-K+-2Cl− cotransporter (Miller & Edelman, 1990; Joseph & Miller, 1991). At light onset, a decrease in subretinal K+ concentration, originating from a change in photoreceptor activity, causes an increase in the efflux of K+ through the apical K+ channels, leading to the reversal of net K+ transport from absorption to secretion (Bialek & Miller, 1994). In patch-clamp studies on RPE cells isolated from a variety of vertebrate species, we have shown that the predominant conductance in the physiological voltage range is an inwardly rectifying K+ (Kir) conductance (Hughes & Steinberg, 1990; Hughes & Takahira, 1996, 1998). The inward rectification of this K+ conductance is relatively weak, such that it supports substantial outward K+ current at voltages positive to the K+ equilibrium potential. This conductance has several remarkable properties, including an inverse dependence on extracellular K+ concentration (Segawa & Hughes, 1994; Hughes & Takahira, 1996) and an intracellular Mg-ATP requirement for sustained activity (Hughes & Takahira, 1998). Blocker sensitivity studies on the intact RPE sheet preparation indicate that these Kir channels underlie that apical membrane K+ conductance (Hughes et al. 1995a). In the mid-1990s, expressional cloning of the inwardly rectifying K+ channels ROMK1 (Ho et al. 1993), IRK1 (Kubo et al. 1993) and GIRK (Kofuji et al. 1995) established the existence of a new gene family distinct from the voltage-gated K+ channel family. Since then, several other members of the Kir channel family have been identified, increasing the number of members to 15 (Reimann & Ashcroft, 1999). The most recent addition is Kir7.1, an inwardly rectifying K+ channel with several novel properties, including a macroscopic conductance with low dependence on extracellular K+ concentration ([K+]o) (Doring et al. 1998; Krapivinsky et al. 1998), a low unitary conductance estimated to be ≈50 fS (Krapivinsky et al. 1998), and an unusually large Rb+-to-K+ conductance ratio (Wischmeyer et al. 2000). Kir7.1 expression has been reported in certain epithelia such as choroid plexus and small intestine, as well as in stomach, kidney, thyroid follicular cells, brain, spinal chord and testis (Doring et al. 1998; Krapivinsky et al. 1998; Partiseti et al. 1998; Nakamura et al. 1999, 2000). The capacity of this channel to pass large outward K+ currents makes it well suited to function in epithelial ion transport processes (Doring et al. 1998). In this study, we have cloned bovine Kir7.1 from a subtracted RPE cDNA library (Chang et al. 1997, 1999), obtained its human orthologue and confirmed its expression in the RPE by Northern blot analysis. Furthermore, we have compared the permeation properties of the native Kir channel in freshly dissociated bovine RPE cells to those of cloned Kir7.1 channels expressed in Xenopus oocytes and find that they are nearly identical. Some of these results have been published in abstract form (Shimura et al. 1999; Yuan et al. 2000).