Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K+ > Rb+, NH4 +, Tl+ ≫ Cs+, Na+, Li+) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl+ > K+ > Rb+ > NH4 + ≫ Na+, Li+). Determination of reversal potentials with submillivolt accuracy shows that K+ is over 150-fold more permeant than Na+. Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 100–1,000 mM. These properties are analogous to those seen in many eukaryotic K+ channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K+ channels.

Basic electrophysiological properties of the KcsA K+ channel were examined in planar lipid bilayer membranes. The channel displays open-state rectification and weakly voltage-dependent gating. Tetraethylammonium blocking affinity depends on the side of the bilayer to which the blocker is added. Addition of Na+ to the trans chamber causes block of open-channel current, while addition to the cis side has no effect. Most striking is the activation of KcsA by protons; channel activity is observed only when the trans bilayer chamber is at low pH. To ascertain which side of the channel faces which chamber, residues with structurally known locations were mapped to defined sides of the bilayer. Mutation of Y82, an external residue, results in changes in tetraethylammonium affinity exclusively from the cis side. Channels with cysteine residues substituted at externally exposed Y82 or internally exposed Q119 are functionally modified by methanethiosulfonate reagents from the cis or trans chambers, respectively. Block by charybdotoxin, known to bind to the channel's external mouth, is observed only when the toxin is added to the cis side of channels mutated to be toxin sensitive. These results demonstrate unambiguously that the protonation sites linked to gating are on the intracellular portion of the KcsA protein.

In the inner ear, sensory hair cells not only detect but also amplify the softest sounds, allowing us to hear over an extraordinarily wide intensity range. This amplification is frequency specific, giving rise to exquisite frequency discrimination. Hair cells detect sounds with their mechanotransduction apparatus, which is only now being dissected molecularly. Signal detection is not the only role of this molecular network; amplification of low-amplitude signals by hair bundles seems to be universal in hair cells. "Fast adaptation," the rapid closure of transduction channels following a mechanical stimulus, appears to be intimately involved in bundle-based amplification.

Localization of mechanotransduction in sensory hair cells to hair bundles requires selective targeting of essential proteins to specific locations. Isoform 2 of the plasma-membrane Ca 2ϩ -ATPase (PMCA2), required for hearing and balance, is found exclusively in hair bundles. We determined the contribution of splicing at the two major splicing sites (A and C) to hair-cell targeting of PMCA2. When PMCA2 isoforms were immunoprecipitated from purified hair bundles of rat utricle, 2w was the only site A variant detected; moreover, immunocytochemistry for 2w in rat vestibular and cochlear tissues indicated that this splice form was located solely in bundles. To demonstrate the necessity of the 2w sequence, we transfected hair cells with PMCA2 containing different variants at splice sites A and C. Although native hair bundles exclusively use the 2a form at splice-site C, epitope-tagged PMCA2w/a and PMCA2w/b were both concentrated in bundles, indicating that site C is not involved in bundle targeting. In contrast, PMCA2z/a was excluded from bundles and was instead targeted to the basolateral plasma membrane. Bundle-specific targeting of PMCA2w/a tagged with green fluorescent protein (GFP) was diminished, suggesting that GFP interfered with splice-site A. Together, these data demonstrate that PMCA2w/a is the hairbundle isoform of PMCA in rat hair cells and that 2w targets PMCA2 to bundles. The 2w sequence is thus the first targeting signal identified for a hair-bundle membrane protein; moreover, the striking distribution of inner-ear PMCA isoforms dictated by selective targeting suggests a critical functional role for segregated pathways of Ca 2ϩ transport.

Platelet activation and aggregation are mediated by thrombin cleavage of the exodomain of the PAR1 receptor. The specificity of thrombin for PAR1 is enhanced by binding to a hirudin-like region (Hir) located in the receptor exodomain. Here, we examine the mechanism of thrombin-PAR1 recognition and cleavage by steadystate kinetic measurements using soluble PAR1 N-terminal exodomains. We determined that the primary role of the PAR1 Hir sequence is to reduce the kinetic barriers to formation of the docked thrombin-PAR1 complex rather than to form high affinity ground-state interactions. In addition, the exosite I-bound Hir motif facilitates the productive interaction of the PAR1 38 LDPR/SFL 44 sequence with the active site of thrombin. This locking process is the most energetically unfavorable step of the overall reaction. The subsequent irreversible steps of peptide bond cleavage are rapid and allosterically enhanced by the presence of the docked Hir sequence. Furthermore, the C-terminal exodomain product of thrombin cleavage, corresponding to the activated receptor, binds tightly to thrombin. This would suggest that an additional role of the Hir sequence in the thrombin-activated receptor is to sequester thrombin to the platelet surface and modulate cleavage of other platelet receptors such as the PAR4 thrombin receptor, which lacks a functional Hir sequence. Cellular responses to thrombin are regulated by a novel class of seven transmembrane protease-activated receptors (PARs). 1 PAR1 plays an important role in platelet activation and blood coagulation and has been implicated in the pathological processes leading to heart disease and stroke (1). PAR1 and PAR4 mediate platelet aggregation in humans, whereas PAR3 and PAR4 are responsible for platelet aggregation in mice (2-4). PAR1 and PAR4 have markedly different kinetics of activation by thrombin that contribute to their distinct roles in signaling in human platelets (5). PAR2 is a trypsin/tryptase receptor that is not cleaved by thrombin (6, 7 (Hir), that resembles the C-tail of the leech anti-coagulant protein, hirudin. The PAR1 Hir sequence interacts with exosite I of thrombin as indicated by x-ray structural analysis (10). Mutagenesis studies indicate that the presence of the Hir sequence in the PAR1 exodomain confers significant enhancements in the efficiency of thrombin cleavage (11-13). In analogy to the dual interactions of hirudin and serpins with thrombin (14, 15), it is possible that the Hir sequence may increase the probability that productive orientations occur at the active site by anchoring the LDPR region and by an induced fit mechanism. Thus, PAR4, which lacks a functional Hir sequence (16), is activated 20-to 70-fold slower by thrombin than PAR1 on human platelets (5).An unusual feature of thrombin-PAR interactions is that stoichiometric amounts of thrombin, rather than catalytic amounts, are required for platelet activation (5). This raises the possibility that the low turnover of thrombin could be due to tight binding and a slow off-rate fro...

Hair cells of the inner ear transduce mechanical stimuli like sound or head movements into electrical signals, which are propagated to the central nervous system. The hair-cell mechanotransduction channel remains unidentified. We tested whether three transient receptor channel (TRP) family members, TRPV6, TRPM6 and TRPM7, were necessary for transduction. TRPV6 interacted with USH1C (harmonin), a scaffolding protein that participates in transduction. Using a cysteine-substitution knock-in mouse line and methanethiosulfonate (MTS) reagents selective for this allele, we found that inhibition of TRPV6 had no effect on transduction in mouse cochlear hair cells. TRPM6 and TRPM7 each interacted with the tip-link component PCDH15 in cultured eukaryotic cells, which suggested they might be part of the transduction complex. Cochlear hair cell transduction was not affected by manipulations of Mg2+, however, which normally perturbs TRPM6 and TRPM7. To definitively examine the role of these two channels in transduction, we showed that deletion of either or both of their genes selectively in hair cells had no effect on auditory function. We suggest that TRPV6, TRPM6 and TRPM7 are unlikely to be the pore-forming subunit of the hair-cell transduction channel.

In this issue, we honor the legacy of David Hubel and Torsten Wiesel, whose pioneering work transformed the field of visual neuroscience. From their early characterization of neuronal response properties in primary visual cortex to their analysis of how experience impacts the development of the visual system, the work of Hubel and Wiesel revealed fundamental insights into cortical architecture, function, and plasticity. The collection of reviews in this issue was inspired by the 50 th anniversary of their landmark paper ''Receptive fields, binocular interaction and functional architecture in the cat's visual cortex,'' published in the Journal of Physiology in 1962 (Hubel and Wiesel, 1962). While the functional and organizational principles laid out in this paper certainly set the stage for a host of subsequent studies in the visual system, its reach has extended far beyond V1. It is a true ''classic'' in neuroscience and has served not only to guide work in the visual system but also to inspire any neuroscientist seeking to understand how the activity of individual neurons can give rise to perception and behavior.

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