Brad S. Rothberg

STIM proteins: dynamic calcium signal transducers

Stromal interaction molecule (STIM) proteins function in cells as dynamic coordinators of cellular calcium (Ca2+) signals. Spanning the endoplasmic reticulum (ER) membrane, they sense tiny changes in the levels of Ca2+ stored within the ER lumen. As ER Ca2+ is released to generate primary Ca2+ signals, STIM proteins undergo an intricate activation reaction and rapidly translocate into junctions formed between the ER and the plasma membrane. There, STIM proteins tether and activate the highly Ca2+-selective Orai channels to mediate finely controlled Ca2+ signals and to homeostatically balance cellular Ca2+. Details are emerging on the remarkable organization within these STIM-induced junctional microdomains and the identification of new regulators and alternative target proteins for STIM.

MICU1 is an Essential Gatekeeper for MCU-Mediated Mitochondrial Ca2+ Uptake That Regulates Cell Survival

Mitochondrial Ca(2+) (Ca(2+)(m)) uptake is mediated by an inner membrane Ca(2+) channel called the uniporter. Ca(2+) uptake is driven by the considerable voltage present across the inner membrane (ΔΨ(m)) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca(2+) concentration is maintained five to six orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here, we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca(2+)](m) under basal conditions. In its absence, mitochondria become constitutively loaded with Ca(2+), triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca(2+) threshold for Ca(2+)(m) uptake without affecting the kinetic properties of MCU-mediated Ca(2+) uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca(2+)(m) uptake that is essential to prevent [Ca(2+)](m) overload and associated stress.

Mechanism of β4 Subunit Modulation of BK Channels

Large-conductance (BK-type) Ca2+-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca2+. BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (β1–β4). Biophysical characterization has shown that the β4 subunit confers properties of the so-called “type II” BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the β4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca2+ sensitivity. Specifically, channel activity at low Ca2+ is inhibited, while at high Ca2+, activity is enhanced. The goal of this study is to understand the mechanism underlying β4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that β4s most profound effect is a decrease in Po (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, β4 promotes channel opening by increasing voltage dependence of Po-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of β4 on BK channels. β4 reduces channel opening by decreasing the intrinsic gating equilibrium (L0), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, β4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vho) to more negative membrane potentials. The consequence is that β4 causes a net positive shift of the G-V relationship (relative to α subunit alone) at low calcium. At higher calcium, the contribution by Vho and an increase in allosteric coupling to Ca2+ binding (C) promotes a negative G-V shift of α+β4 channels as compared to α subunits alone. This manner of modulation predicts that type II BK channels are downregulated by β4 at resting voltages through effects on L0. However, β4 confers a compensatory effect on voltage sensor activation that increases channel opening during depolarization.

Distinct Orai-coupling domains in STIM1 and STIM2 define the Orai-activating site

STIM1 and STIM2 are widely expressed endoplasmic reticulum (ER) Ca(2+) sensor proteins able to translocate within the ER membrane to physically couple with and gate plasma membrane Orai Ca(2+) channels. Although they are structurally similar, we reveal critical differences in the function of the short STIM-Orai-activating regions (SOAR) of STIM1 and STIM2. We narrow these differences in Orai1 gating to a strategically exposed phenylalanine residue (Phe-394) in SOAR1, which in SOAR2 is substituted by a leucine residue. Remarkably, in full-length STIM1, replacement of Phe-394 with the dimensionally similar but polar histidine head group prevents both Orai1 binding and gating, creating an Orai1 non-agonist. Thus, this residue is critical in tuning the efficacy of Orai activation. While STIM1 is a full Orai1-agonist, leucine-replacement of this crucial residue in STIM2 endows it with partial agonist properties, which may be critical for limiting Orai1 activation stemming from its enhanced sensitivity to store-depletion.

Movements near the gate of a hyperpolarization-activated cation channel.

Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Like the related depolarization-activated K+ channels (Kv channels), HCN channels use an intracellular activation gate to regulate access to an inner cavity, lined by the S6 transmembrane regions, which leads to the selectivity filter near the extracellular surface. Here we describe two types of metal interactions with substituted cysteines in the S6, which alter the voltage-controlled movements of the gate. At one position (L466), substitution of cysteine in all four subunits allows Cd2+ ions at nanomolar concentration to stabilize the open state (a “lock-open” effect). This effect depends on native histidines at a nearby position (H462); the lock-open effect can be abolished by changing the histidines to tyrosines, or enhanced by changing them to cysteines. Unlike a similar effect in Kv channels, this effect depends on a Cd2+ bridge between 462 and 466 in the same subunit. Cysteine substitution at another position (Q468) produces two effects of Cd2+: both a lock-open effect and a dramatic slowing of channel activation—a “lock-closed” effect. The two effects can be separated, because the lock-open effect depends on the histidine at position 462. The novel lock-closed effect results from stabilization of the closed state by the binding of up to four Cd2+ ions. During the opening conformational change, the S6 apparently moves from one position in which the 468C cysteines can bind four Cd2+ ions, possibly as a cluster of cysteines and cadmium ions near the central axis of the pore, to another position (or flexible range of positions) where either 466C or 468C can bind Cd2+ in association with the histidine at 462.

Orai channel pore properties and gating by STIM: implications from the Orai crystal structure.

Crystallographic data enable predictions regarding gating of the store-operated calcium entry channel. The Orai channels are unusual, yet prominent, calcium (Ca2+) signal mediators in most cell types. Orai proteins are structurally unique, having little sequence homology with other ion channels. They are also functionally unique with exceedingly high selectivity for Ca2+, mediating both short-term Ca2+ homeostasis and long-term Ca2+ signals important for transcriptional control. Operating in the plasma membrane (PM), Orai channel regulation is unprecedented among ion channels; channel gating occurs through an elaborate intermembrane coupling with stromal interaction molecule (STIM) proteins in the endoplasmic reticulum (ER). STIM proteins function as sensors of Ca2+ stored in the ER lumen and translocate into ER-PM junctions to tether and activate Orai channels when ER Ca2+ concentration decreases. Crystallization studies reveal an unexpected hexameric structure for the Orai channel and provide important insights into the pore architecture, the structural basis of its unusual cation selectivity, and how channel gating occurs through its coupling with STIM proteins.

Mechanism of Increased BK Channel Activation from a Channel Mutation that Causes Epilepsy

Concerted depolarization and Ca2+ rise during neuronal action potentials activate large-conductance Ca2+- and voltage-dependent K+ (BK) channels, whose robust K+ currents increase the rate of action potential repolarization. Gain-of-function BK channels in mouse knockout of the inhibitory β4 subunit and in a human mutation (αD434G) have been linked to epilepsy. Here, we investigate mechanisms underlying the gain-of-function effects of the equivalent mouse mutation (αD369G), its modulation by the β4 subunit, and potential consequences of the mutation on BK currents during action potentials. Kinetic analysis in the context of the Horrigan-Aldrich allosteric gating model revealed that changes in intrinsic and Ca2+-dependent gating largely account for the gain-of-function effects. D369G causes a greater than twofold increase in the closed-to-open equilibrium constant (6.6e−7→1.65e−6) and an approximate twofold decrease in Ca2+-dissociation constants (closed channel: 11.3→5.2 µM; open channel: 0.92→0.54 µM). The β4 subunit inhibits mutant channels through a slowing of activation kinetics. In physiological recording solutions, we established the Ca2+ dependence of current recruitment during action potential–shaped stimuli. D369G and β4 have opposing effects on BK current recruitment, where D369G reduces and β4 increases K1/2 (K1/2 μM: αWT 13.7, αD369G 6.3, αWT/β4 24.8, and αD369G/β4 15.0). Collectively, our results suggest that the D369G enhancement of intrinsic gating and Ca2+ binding underlies greater contributions of BK current in the sharpening of action potentials for both α and α/β4 channels.

A Role for the S0 Transmembrane Segment in Voltage-dependent Gating of BK Channels

BK (Maxi-K) channel activity is allosterically regulated by a Ca2+ sensor, formed primarily by the channels large cytoplasmic carboxyl tail segment, and a voltage sensor, formed by its transmembrane helices. As with other voltage-gated K channels, voltage sensing in the BK channel is accomplished through interactions of the S1–S4 transmembrane segments with the electric field. However, the BK channel is unique in that it contains an additional amino-terminal transmembrane segment, S0, which is important in the functional interaction between BK channel α and β subunits. In this study, we used perturbation mutagenesis to analyze the role of S0 in channel gating. Single residues in the S0 region of the BK channel were substituted with tryptophan to give a large change in side chain volume; native tryptophans in S0 were substituted with alanine. The effects of the mutations on voltage- and Ca2+-dependent gating were quantified using patch-clamp electrophysiology. Three of the S0 mutants (F25W, L26W, and S29W) showed especially large shifts in their conductance–voltage (G-V) relations along the voltage axis compared to wild type. The G-V shifts for these mutants persisted at nominally 0 Ca2+, suggesting that these effects cannot arise simply from altered Ca2+ sensitivity. The basal open probabilities for these mutants at hyperpolarized voltages (where voltage sensor activation is minimal) were similar to wild type, suggesting that these mutations may primarily perturb voltage sensor function. Further analysis using the dual allosteric model for BK channel gating showed that the major effects of the F25W, L26W, and S29W mutations could be accounted for primarily by decreasing the equilibrium constant for voltage sensor movement. We conclude that S0 may make functional contact with other transmembrane regions of the BK channel to modulate the equilibrium between resting and active states of the channels voltage sensor.

High Ca2+ concentrations induce a low activity mode and reveal Ca2(+)-independent long shut intervals in BK channels from rat muscle.

1. Large‐conductance calcium‐activated K+ channels (BK channels) often display long closed intervals at higher levels of Ca2+. To gain further insight into possible mechanisms for these intervals, currents were recorded from single BK channels, using the patch clamp technique, from patches of membrane excised from primary cultures of rat skeletal muscle. 2. High intracellular calcium concentrations ([Ca2+]i; 10‐1000 microM) induced a low activity mode and revealed isolated long shut intervals. Neither of these phenomena were due to the Ba2+ that typically contaminates reagent grade salts. 3. The low activity mode was characterized by typically single brief open intervals with mean durations of 0.1 ms, separated by long shut intervals with mean durations of 100 ms. The very low open probability of about 0.001 during the low activity mode would make a sojourn to this mode functionally equivalent to a sojourn to an inactive state. The durations of sojourns in the low activity mode were exponentially distributed, with the mean durations ranging from about 1 s in 10 microM Ca(i)2+, to 4.5 s in 1000 microM Ca(i)2+. With increased filtering, the brief open intervals would escape detection so that a sojourn to the low activity mode would appear as a single shut interval. A typical channel spent less than 5% of its time in the low activity mode for [Ca2+]i < 10 microM. This increased to about 30% for [Ca2+]i > 100‐1000 microM. A kinetic model with three closed states and two open states could approximate the gating of the low activity mode. 4. The isolated long shut intervals were not from the low activity mode, suggesting a different underlying mechanism. Their frequency of occurrence of about 0.3 s‐1 did not increase with increasing [Ca2+]i, indicating that they did not arise from a slow Ca2+ block. Their durations were exponentially distributed, with a mean of 127 ms, which was independent of [Ca2+]i, suggesting that a single Ca(2+)‐independent closed state or block underlies the isolated long shut intervals. At higher [Ca2+]i, up to 60% of the shut time could be spent in the isolated long shut intervals. 5. These observations suggest that activation of BK channels by high [Ca2+]i can be limited by sojourns to a low activity mode and also by isolated long shut intervals, two additional phenomena that will have to be accounted for in the gating of BK channels.

The BK channel: a vital link between cellular calcium and electrical signaling

Large-conductance Ca2+-activated K+ channels (BK channels) constitute an key physiological link between cellular Ca2+ signaling and electrical signaling at the plasma membrane. Thus these channels are critical to the control of action potential firing and neurotransmitter release in several types of neurons, as well as the dynamic control of smooth muscle tone in resistance arteries, airway, and bladder. Recent advances in our understanding of K+ channel structure and function have led to new insight toward the molecular mechanisms of opening and closing (gating) of these channels. Here we will focus on mechanisms of BK channel gating by Ca2+, transmembrane voltage, and auxiliary subunit proteins.