-activated Chloride Channels Go Molecular

With the sequencing of the human genome it turned out that many of the genetically identified genes have an unknown function and one of the major problems of biology is the “annotation” of the genome. But the reverse problem persists, i.e., that for many functionally well-defined proteins the coding gene has not been identified. This reverse problem is difficult to tackle because the molecular tools that help for the annotation (for example knock-out, heterologous expression, localization) cannot be applied. For ion channels in particular, biochemical approaches are also hampered by the low protein abundance. These are well-known problems for two different classes of Cl ion channels: “swelling”-activated and Ca 2 -activated channels. The latter type of Cl channel is involved in epithelial salt transport, in the regulation of the membrane potential of smooth and heart muscle cells, in the block of polyspermi in oocytes, in the amplification of the receptor potential in odorant receptor cells, and probably many other physiological processes (for review see Jentsch et al., 2002). Despite considerable efforts in the past years these physiologically important ion channels have been notoriously difficult to identify at the molecular level. In this issue of the Journal of General Physiology , Criss Hartzell’s group (Qu et al., 2004) makes a significant step forward to fill this knowledge gap for Ca 2 -activated Cl channels. In short, the authors demonstrate that the mouse bestrophin 2 protein (mBest2) by itself forms a Ca 2 -dependent Cl channel in heterologous expression systems and they analyze in great detail the functional properties of this channel. Bestrophin 1 was identified 6 yr ago as the gene product of the VMD2 gene, mutations of which cause Best vitelliform macular dystrophy (or Best disease) (Marquardt et al., 1998; Petrukhin et al., 1998). The disease is characterized by a dominant, early onset macular degeneration associated with an accumulation of lipofuscin-like material at the level of the retinal pigment epithelium (RPE). A total of four homologues have been identified in humans and similar proteins are also found in many other species like, e.g., Xenopus laevis (Qu et al., 2003). In Caenorhabditis elegans , 24 members of the family are found. Initially, their function was completely unknown, but the presence of several hydrophobic segments suggested that they might be transport proteins. Such a role could explain the macular dystrophy as a membrane transport defect in the RPE. Indeed, several recent papers reported the appearance of a Cl conductance when each of the four human bestrophins or their Xenopus homologues were heterologously expressed in HEK-293 cells (Sun et al., 2002; Tsunenari et al., 2003; Qu et al., 2003). Currents induced by the expression of hBest1 described initially by Sun et al. (2002) were sensitive to intracellular [Ca 2 ]. So why are the results of Qu et al. (2004) so significant if it was already known that bestrophins are Cl channels? The fact is that the earlier reports did not actually prove that the bestrophin proteins are a structural part of the pore-containing channel. They were equally compatible with the hypothesis that bestrophins up-regulate a Cl current carried by another channel protein. This distinction might seem like hairsplitting, but in the Cl channel field such false positive candidates have plagued researchers more than once (for reviews see Clapham, 1998; Jentsch et al., 2002). Based on the earlier results of Tsunenari et al. (2003), it was relatively accepted that bestrophins are located (at least partially) in the plasma membrane, and that they probably directly interact with (or are themselves) the pore protein. These authors could abolish an inhibitory effect of sulfhydryl reactive MSET by removing specific cysteines. This, together with further biochemical experiments, allowed a tentative assignment of the transmembrane topology of bestrophins. An additional, and strong, argument for bestrophin being part of the pore-forming protein is provided now by Qu et al. (2004): Currents induced by wild-type mBest2 are blocked by extracellular SCN ions with an IC 50 12 mM. The authors identified an amino acid (S79) that practically abolished this block when mutated to cysteine. In addition, the same mutation also altered the relative SCN /Cl permeability ratio and reduced block by DIDS. These results strongly suggest that the residue S79 directly participates in the ion conduction process and thus that the bestrophin protein is a structural component of the ion conducting pore. Qu et al. (2004) perform further detailed experiments to characterize the channel. For example, they determine T h e Jo u rn al o f G en er al P h ys io lo g y