Chris S. Gandhi

Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel.

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.

Molecular Models of Voltage Sensing

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

The orientation and molecular movement of a k(+) channel voltage-sensing domain.

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.

Cu(II) inhibition of the proton translocation machinery of the influenza a virus M2 protein

The homotetrameric M2 integral membrane protein of influenza virus forms a proton-selective ion channel. An essential histidine residue (His-37) in the M2 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 M2protein were recorded when the solution bathing the oocytes contained various transition metals. Membrane currents were strongly and reversibly inhibited by Cu2+ 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 ∼2 μm) that lies deep within the pore and shows rather slow-binding kinetics (k on = 18.6 ± 0.9m −1 s−1). The pH dependence of the interaction with the high affinity Cu2+-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 Cu2+ could be prevented by prior application of the reversible blocker of M2 channel activity, BL-1743, providing further support for the location of the site within the pore region of M2. 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 Cu2+. Binding of Cu2+ 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 Cu2+ and was only partially inhibited by 1 mm Ni2+, Pt2+, and Zn2+. These data are discussed in terms of the functional role of His-37 in the mechanism of proton translocation through the channel.

Structure of a tetrameric MscL in an expanded intermediate state

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 osmoregulation. 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 down-shock. Among them, MscL has been intensively studied since the original identification and characterization 15 years ago. MscL is reversibly and directly gated by changes in membrane tension. In the open state, MscL forms a non-selective 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 represents a closed-state or non-conducting conformation. MscL has a complex gating behaviour; it exhibits several intermediates between the closed and open states, including one putative non-conductive expanded state and at least three sub-conducting states. Although our understanding of the closed and open 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 carboxy-terminal truncation mutant (Δ95–120) of MscL from Staphylococcus aureus (SaMscL(CΔ26)) at 3.8 Å resolution. Notably, SaMscL(CΔ26) forms a tetrameric channel with both transmembrane helices tilted away from the membrane normal at angles close to that inferred for the open state, probably corresponding to a non-conductive but partially expanded intermediate state.

Reconstructing Voltage Sensor–Pore Interaction from a Fluorescence Scan of a Voltage-Gated K+ Channel

X-ray crystallography has made considerable recent progress in providing static structures of ion channels. Here we describe a complementary method-systematic fluorescence scanning-that reveals the structural dynamics of a channel. Local protein motion was measured from changes in the fluorescent intensity of a fluorophore attached at one of 37 positions in the pore domain and in the S4 voltage sensor of the Shaker K+ channel. The local rearrangements that accompany activation and slow inactivation were mapped onto the homologous structure of the KcsA channel and onto models of S4. The results place clear constraints on S4 location, voltage-dependent movement, and the mechanism of coupling of S4 motion to the operation of the slow inactivation gate in the pore domain.

The RCK2 domain of the human BKCa channel is a calcium sensor

Large conductance voltage and Ca2+-dependent K+ channels (BKCa) are activated by both membrane depolarization and intracellular Ca2+. Recent studies on bacterial channels have proposed that a Ca2+-induced conformational change within specialized regulators of K+ conductance (RCK) domains is responsible for channel gating. Each pore-forming α 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 Ca2+ sensitivity, possibly participating in the formation of a Ca2+-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 Ca2+-binding site within this region is not yet established. Here, we present a structure-based alignment of the C terminus of BKCa and prokaryotic RCK domains that reveal the location of a second RCK domain in human BKCa channels (hSloRCK2). hSloRCK2 includes a high-affinity Ca2+-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 Ca2+-induced change in conformation, associated with an α-to-β structural transition. We also show that the Ca bowl is an essential element for the Ca2+-induced rearrangement of hSloRCK2. We speculate that the molecular rearrangements of RCK2 likely underlie the Ca2+-dependent gating mechanism of BKCa channels. A structural model of the heterodimeric complex of hSloRCK1 and hSloRCK2 domains is discussed.

The RCK1 domain of the human BKCa channel transduces Ca2+ binding into structural rearrangements

Large-conductance voltage- and Ca2+-activated K+ (BKCa) channels play a fundamental role in cellular function by integrating information from their voltage and Ca2+ sensors to control membrane potential and Ca2+ homeostasis. The molecular mechanism of Ca2+-dependent regulation of BKCa channels is unknown, but likely relies on the operation of two cytosolic domains, regulator of K+ conductance (RCK)1 and RCK2. Using solution-based investigations, we demonstrate that the purified BKCa RCK1 domain adopts an α/β fold, binds Ca2+, and assembles into an octameric superstructure similar to prokaryotic RCK domains. Results from steady-state and time-resolved spectroscopy reveal Ca2+-induced conformational changes in physiologically relevant [Ca2+]. The neutralization of residues known to be involved in high-affinity Ca2+ sensing (D362 and D367) prevented Ca2+-induced structural transitions in RCK1 but did not abolish Ca2+ binding. We provide evidence that the RCK1 domain is a high-affinity Ca2+ sensor that transduces Ca2+ binding into structural rearrangements, likely representing elementary steps in the Ca2+-dependent activation of human BKCa channels.

Single-Cell Census of Mechanosensitive Channels in Living Bacteria

Bacteria are subjected to a host of different environmental stresses. One such insult occurs when cells encounter changes in the osmolarity of the surrounding media resulting in an osmotic shock. In recent years, a great deal has been learned about mechanosensitive (MS) channels which are thought to provide osmoprotection in these circumstances by opening emergency release valves in response to membrane tension. However, even the most elementary physiological parameters such as the number of MS channels per cell, how MS channel expression levels influence the physiological response of the cells, and how this mean number of channels varies from cell to cell remain unanswered. In this paper, we make a detailed quantitative study of the expression of the mechanosensitive channel of large conductance (MscL) in different media and at various stages in the growth history of bacterial cultures. Using both quantitative fluorescence microscopy and quantitative Western blots our study complements earlier electrophysiology-based estimates and results in the following key insights: i) the mean number of channels per cell is much higher than previously estimated, ii) measurement of the single-cell distributions of such channels reveals marked variability from cell to cell and iii) the mean number of channels varies under different environmental conditions. The regulation of MscL expression displays rich behaviors that depend strongly on culturing conditions and stress factors, which may give clues to the physiological role of MscL. The number of stress-induced MscL channels and the associated variability have far reaching implications for the in vivo response of the channels and for modeling of this response. As shown by numerous biophysical models, both the number of such channels and their variability can impact many physiological processes including osmoprotection, channel gating probability, and channel clustering.

Metal-driven Operation of the Human Large-conductance Voltage- and Ca2+-dependent Potassium Channel (BK) Gating Ring Apparatus

Large-conductance voltage- and Ca2+-dependent K+ (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca2+ homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the “gating ring”), which confers sensitivity to intracellular Ca2+ and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca2+- and Mg2+-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba2+-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca2+-binding site (the “calcium bowl”) reduced the Ca2+ and abolished the Mg2+ dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca2+-binding site and that Mg2+ can bind to the calcium bowl with less affinity than Ca2+. Dynamic light scattering analysis revealed a reversible Ca2+-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel.