The ability to differentiate between ions is a property of ion channels that is crucial for their biological functions. However, the fundamental structural features that define anion selectivity and distinguish anion-permeable from cation-permeable channels are poorly understood. Voltage-gated chloride (Cl-) channels belonging to the ClC family are ubiquitous and have been predicted to play important roles in many diverse physiological and pathophysiological processes. We have identified regions of a human skeletal muscle ClC isoform that contribute to formation of its anion-selective conduction pathway. A core structural element (P1 region) of the ClC channel pore spans an accessibility barrier between the internal and external milieu, and contains an evolutionarily conserved sequence motif: GKxGPxxH. Neighbouring sequences in the third and fifth transmembrane segments also contribute to isoform-specific differences in anion selectivity. The conserved motif in the Cl- channel P1 region may constitute a 'signature' sequence for an anion-selective ion pore by analogy with the homologous GYG sequence that is essential for selectivity in voltage-gated potassium ion (K+) channel pores.

Voltage-dependent gating was investigated in a recombinant human skeletal muscle Cl- channel, hCIC-1, heterologously expressed in human embryonic kidney (HEK-293) cells. Gating was found to be mediated by two qualitatively distinct processes. One gating step operates on a microsecond time scale and involves the rapid rearrangement of two identical intramembranous voltage sensors, each consisting of a single titratable residue. The second process occurs on a millisecond time scale and is due to a blocking-unblocking reaction mediated by a cytoplasmic gate that interacts with the ion pore of the channel. These results illustrate a rather simple structural basis for voltage sensing that has evolved in skeletal muscle Cl- channels and provides evidence for the existence of a cytoplasmic gating mechanism in an anion channel analogous to the "ball and chain" mechanism of voltage-gated cation channels.

Voltage-gated anion channels are present in almost every living cell and have many physiological functions. Recently, a novel gene family encoding voltage-gated chloride channels, the ClC family, was identified. The knowledge of primary amino acid sequences has allowed for the study of these anion channels in heterologous expression systems and made possible the combination of site-directed mutagenesis and high-resolution electrophysiological measurements as a means of gaining insights into the molecular basis of channel function. This review focuses on one particular aspect of chloride channel function, the selective transport of anions through biological membranes. I will describe recent experiments using a combination of cellular electrophysiology, molecular genetics, and recombinant DNA technology to study the molecular basis of ion permeation and selection in ClC-type chloride channels. These novel tools have provided new insights into basic mechanisms underlying the function of these biologically important channels.

Recently, two groups of rhodopsin genes were identified in large double-stranded DNA viruses. The structure and function of viral rhodopsins are unknown. We present functional characterization and high-resolution structure of an Organic Lake Phycodnavirus rhodopsin II (OLPVRII) of group 2. It forms a pentamer, with a symmetrical, bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of 5 arginines, whereas 5 phenylalanines form a hydrophobic barrier in its exit. The proton donor E42 is placed in the helix B. The structure is unique among the known rhodopsins. Structural and functional data and molecular dynamics suggest that OLPVRII might be a light-gated pentameric ion channel analogous to pentameric ligand-gated ion channels, however, future patch clamp experiments should prove this directly. The data shed light on a fundamentally distinct branch of rhodopsins and may contribute to the understanding of virus-host interactions in ecologically important marine protists.