The M 2 protein from inf luenza A virus forms proton-selective channels that are essential to viral function and are the target of the drug amantadine. Cys scanning was used to generate a series of mutants with successive substitutions in the transmembrane segment of the protein, and the mutants were expressed in Xenopus laevis oocytes. The effect of the mutations on reversal potential, ion currents, and amantadine resistance were measured. Fourier analysis revealed a periodicity consistent with a four-stranded coiled coil or helical bundle. A three-dimensional model of this structure suggests a possible mechanism for the proton selectivity of the M 2 channel of inf luenza virus.Ion channels are responsible for the rapid and efficient conduction of ions across phospholipid bilayers. They are generally highly selective for their permeant ions, and are gated by voltage or ligands (1). Although a number of high-resolution structures are available for hemolysins (2) and porins (3)-channel-like proteins that form large, nonselective poresstructural analysis of more selective ion channel proteins is at an early stage. Sequence analysis and low-resolution diffraction data indicate that their conduction pathways often consist of bundles of ␣-helices (4, 5), but the determination of high-resolution structures of channel proteins has been hampered by their limited availability and large size.M 2 from influenza virus is an essential component of the viral envelope and forms a highly selective, pH-regulated proton channel that is the target of the anti-influenza drug amantadine (6-9). The influenza virus enters cells through internalization into the endocytic pathway, with virus uncoating taking place in endosomal compartments. The M 2 ion channel activity permits protons to enter the virion interior, and this acidification weakens the interactions of the matrix protein (M 1 ) with the ribonucleoprotein core (10). By comparison to the channels of excitable tissues, M 2 is quite small (97 residues) and contains but one hydrophobic stretch of 18 residues believed to form a transmembrane (TM) helix (residues 26-43). A wealth of experimental evidence indicates that the M 2 channel DPL 26 VVAASIIGILHLILWIL 43 D consists of a tetrameric array of parallel, TM peptides with their N termini directed toward the outside of the virus (6-9). A synthetic 25-residue peptide spanning the hydrophobic region forms amantadine-sensitive proton channels, indicating that the determinants for assembly of the channel lie within this TM peptide (11). Further, CD spectroscopy indicates that this peptide forms ␣-helices in membranes (12). Thus, the TM region of the channel appears to consist of a parallel bundle of ␣-helices.Here we describe the use of Cys-scanning mutagenesis (13,17,18) to obtain more detailed information concerning the arrangement of the TM helices within the tetrameric pore. A similar method has been used previously to infer the probable structures of other homo-oligomeric TM proteins, including glycophorin (14) and phosph...

Voltage-gated ion channels underlie the generation of action potentials and trigger neurosecretion and muscle contraction. These channels consist of an inner pore-forming domain, which contains the ion permeation pathway and elements of its gates, together with four voltage-sensing domains, which regulate the gates. To understand the mechanism of voltage sensing it is necessary to define the structure and motion of the S4 segment, the portion of each voltage-sensing domain that moves charged residues across the membrane in response to voltage change. We have addressed this problem by using fluorescence resonance energy transfer as a spectroscopic ruler to determine distances between S4s in the Shaker K+ channel in different gating states. Here we provide evidence consistent with S4 being a tilted helix that twists during activation. We propose that helical twist contributes to the movement of charged side chains across the membrane electric field and that it is involved in coupling voltage sensing to gating.

The ability of cells to sense and respond to mechanical force underlies diverse processes such as touch and hearing in animals, gravitropism in plants, and bacterial osmoregulation1, 2. In bacteria, mechanosensation is mediated by the mechanosensitive channels of large (MscL), small (MscS), potassium-dependent (MscK), and mini (MscM) conductances. These channels act as “emergency relief valves” protecting bacteria from lysis upon acute osmotic downshock3. Among them, MscL has been intensively studied since the original identification and characterization 15 years ago by Kung and co-workers4. MscL is reversibly and directly gated by changes in membrane tension. In the open state, MscL forms a nonselective 3 nS-conductance channel which gates at tensions close to the lytic limit of the bacterial membrane. An earlier crystal structure at 3.5 Å resolution of a pentameric MscL from Mycobacterium tuberculosis (TbMscL) represents a closed-state or nonconducting conformation5, 6. MscL has a complex gating behaviour; it exhibits several intermediates between the closed and open states, including one putative nonconductive expanded state and at least three sub-conducting states7. Although our understanding of the closed5, 6 and open8-10 states of MscL has been increasing, little is known about the structures of the intermediate states despite their importance in elucidating the complete gating process of MscL. Here we present the crystal structure of a truncation mutant (Δ95-120) of MscL from Staphylococcus aureus (SaMscL-CΔ26) at 3.8 Å resolution. Strikingly, SaMscL-CΔ26 forms a tetrameric channel with both transmembrane (TM) helices tilted away from the membrane normal at angles close to that inferred for the open state9, likely corresponding to a nonconductive but partially expanded intermediate state.

Voltage-gated channels operate through the action of a voltage-sensing domain (membrane segments S1-S4) that controls the conformation of gates located in the pore domain (membrane segments S5-S6). Recent structural studies on the bacterial K(v)AP potassium channel have led to a new model of voltage sensing in which S4 lies in the lipid at the channel periphery and moves through the membrane as a unit with a portion of S3. Here we describe accessibility probing and disulfide scanning experiments aimed at determining how well the K(v)AP model describes the Drosophila Shaker potassium channel. We find that the S1-S3 helices have one end that is externally exposed, S3 does not undergo a transmembrane motion, and S4 lies in close apposition to the pore domain in the resting and activated state.

The homotetrameric M 2 integral membrane protein of influenza virus forms a proton-selective ion channel. An essential histidine residue (His-37) in the M 2 transmembrane domain is believed to play an important role in the conduction mechanism of this channel. Also, this residue is believed to form hydrogen-bonded interactions with the ammonium group of the anti-viral compound, amantadine. A molecular model of this channel suggests that the imidazole side chains of His-37 from symmetry-related monomers of the homotetrameric pore converge to form a coordination site for transition metals. Thus, membrane currents of oocytes of Xenopus laevis expressing the M 2 protein were recorded when the solution bathing the oocytes contained various transition metals. Membrane currents were strongly and reversibly inhibited by Cu 2؉ with biphasic reaction kinetics. The biphasic inhibition curves may be explained by a two-site model involving a fast-binding peripheral site with low specificity for divalent metal ions, as well as a high affinity site (K diss ϳ2 M) that lies deep within the pore and shows rather slow-binding kinetics (k on ‫؍‬ 18.6 ؎ 0.9 M ؊1 s ؊1 ). The pH dependence of the interaction with the high affinity Cu 2؉ -binding site parallels the pH dependence of inhibition by amantadine, which has previously been ascribed to protonation of His-37. The voltage dependence of the inhibition at the high affinity site indicates that the binding site lies within the transmembrane region of the pore. Furthermore, the inhibition by Cu 2؉ could be prevented by prior application of the reversible blocker of M 2 channel activity, BL-1743, providing further support for the location of the site within the pore region of M 2 . Finally, substitutions of His-37 by alanine or glycine eliminated the high affinity site and resulted in membrane currents that were only partially inhibited at millimolar concentrations of Cu 2؉ . Binding of Cu 2؉ to the high affinity site resulted in an approximately equal inhibition of both inward and outward currents. The wild-type protein showed very high specificity for Cu 2؉ and was only partially inhibited by 1 mM Ni 2؉ , Pt 2؉ , and Zn 2؉. These data are discussed in terms of the functional role of His-37 in the mechanism of proton translocation through the channel.The M 2 protein of influenza A virus is thought to function as an ion channel that permits protons to enter virus particles during uncoating of virions in endosomes. In addition, in influenza virus-infected cells the M 2 protein causes the equilibration of pH between the acidic lumen of the trans-Golgi network and the cytoplasm (reviewed in Refs. 1 and 2). The M 2 protein contains a 24-residue N-terminal extracellular domain, a single internal hydrophobic domain of 19 residues which acts as a transmembrane domain and forms the pore of the channel, and a 54-residue cytoplasmic tail (3). Chemical cross-linking studies showed the M 2 protein to be minimally a homotetramer (4 -6), and statistical analysis of the ion channel activity of mixed...

The usefulness of fluorescence in studying protein motions derives from its sensitivity, kinetic resolution, and compatibility with both live cells and physiological assays. Recent advances in microscopy and membrane protein purification have permitted the observation of fluorescence changes that accompany the functional transitions of complex eukaryotic membrane proteins. These techniques rely on probes that can clearly report the environmental changes of specific residues, but most commonly available sidechain-reactive probes are not well suited for this purpose. Here, we introduce a red Cys-reactive probe, aminophenoxazone maleimide (APM), designed with improved chemical and spectral properties for reporting protein conformational change. APM is compact, uncharged, and has a short linker between probe and protein, all of which ensure that it can closely track the motions of the side chain to which it is attached. It undergoes large polarity-dependent changes in Stokes shift, as well as large bathochromic shifts in both excitation maximum (from 521 nm in toluene to 598 nm in water) and emission maximum (580 nm to 633 nm). These polaritydependent spectral changes offer a potentially simple means of relating fluorescence to local structure and motion, although they are partially offset by some complicating factors in APM fluorescence. We find that, like a rhodamine maleimide, APM senses the conformational changes underlying voltage sensing in the Shaker potassium channel, and it is superior at a site that shows limited reactivity to the rhodamine. The spectral characteristics of APM can also report subtle differences between aqueous positions in purified preparations of the ␤2 adrenergic receptor.fluorescene ͉ membrane proteins ͉ protein dynamics

Voltage-gated ion channels have always been overachievers. They have the singular distinction of having solved the permeation problem five times over. Not only do they have a central, highly selective pore through which they conduct charged ions, they also have four peripheral "pores" or gating canals through which they conduct the charged portions of their voltage sensors. This trick of protein permeation generates a small gating current as the S4 arginines and lysines move through the electric field of the membrane and ultimately results in channel opening.The membrane-spanning portion of voltage-gated channels contains two classes of functional domains. Four voltage-sensing domains located at the periphery of the tetramer surround a central pore forming domain (Fig. 1 B). The pore domain of the voltage-gated K ϩ (Kv) channels share structural homology with the bacterial KcsA and MthK channels whose crystal structures have been solved (Doyle et al., 1998;Jiang et al., 2002). The pore domain consists of S5, the P-loop, and S6 which constitute the ion permeation pathway, including the selectivity filter and two of the gates (Fig. 1). The voltage-sensing domains, whose crystal structures have yet to be determined, are the subject of this Perspective. The four positively charged S4s, located between S1-S3 and the pore domain, function as voltage sensors. Membrane depolarization drives the positively charged residues of S4 through the gating canal. The movement of these charges through the membrane electric field generates the gating current that precedes channel opening. Below, we explore possible models of voltage sensor structure and motion with the ultimate goal of understanding how voltage-sensor rearrangements drive the pore domain gates to open and close. We begin by outlining eight fundamental experimental observations in the field and then discuss models that could account for these observations.

Large conductance voltage and Ca 2؉ -dependent K ؉ channels (BKCa) are activated by both membrane depolarization and intracellular Ca 2؉ . Recent studies on bacterial channels have proposed that a Ca 2؉ -induced conformational change within specialized regulators of K ؉ conductance (RCK) domains is responsible for channel gating. Each pore-forming ␣ subunit of the homotetrameric BKCa channel is expected to contain two intracellular RCK domains. The first RCK domain in BKCa channels (RCK1) has been shown to contain residues critical for Ca 2؉ sensitivity, possibly participating in the formation of a Ca 2؉ -binding site. The location and structure of the second RCK domain in the BKCa channel (RCK2) is still being examined, and the presence of a high-affinity Ca 2؉ -binding site within this region is not yet established. Here, we present a structure-based alignment of the C terminus of BK Ca and prokaryotic RCK domains that reveal the location of a second RCK domain in human BK Ca channels (hSloRCK2). hSloRCK2 includes a high-affinity Ca 2؉ -binding site (Ca bowl) and contains similar secondary structural elements as the bacterial RCK domains. Using CD spectroscopy, we provide evidence that hSloRCK2 undergoes a Ca 2؉ -induced change in conformation, associated with an ␣-to-␤ structural transition. We also show that the Ca bowl is an essential element for the Ca 2؉ -induced rearrangement of hSloRCK2. We speculate that the molecular rearrangements of RCK2 likely underlie the Ca 2؉ -dependent gating mechanism of BKCa channels. A structural model of the heterodimeric complex of hSloRCK1 and hSloRCK2 domains is discussed.BK channel ͉ circular dichroism ͉ MaxiK ͉ RCK ͉ structural modeling B K Ca channels are formed by the assembly of four identical pore-forming ␣ subunits. They can couple the membrane potential to the intracellular Ca 2ϩ level (1-4), playing critical roles in cell excitability, for example, by controlling smooth muscle tone and neurotransmitter release (1, 5-7). Each BK Ca ␣ subunit possesses a transmembrane voltage sensor (8-10) and two distinct high-affinity Ca 2ϩ sensors (11-15) located within the large intracellular carboxyl terminus. A well studied Ca 2ϩ -binding site corresponds to a C-terminal region that includes five consecutive negatively charged aspartates (D894-D898), christened the ''Ca bowl'' by the Salkoff laboratory (16,17). The Ca bowl binds Ca 2ϩ with high affinity (18-21) and strongly contributes to the channel's Ca 2ϩ sensitivity (18)(19)(20) [supporting information (SI) Fig. 6]. A second high-affinity Ca 2ϩ -sensing region that is impaired by neutralization of two aspartates (D362/D367) (11, 15) or methionine 513 (22) has been identified Ϸ400 aa upstream the Ca bowl.Most likely, these two high-affinity Ca 2ϩ -binding sites form parts of a complex functional domain that converts the free energy of Ca 2ϩ binding into mechanical work to open the channel. Indeed, specialized intracellular motifs regulating the conductance of K ϩ channels (RCK domains) have been recently described in prok...