Riccardo Olcese

Structures and Functions of Calcium Channel β Subunits

Calcium channel β subunits have profound effects on how α1 subunits perform. In this article we summarize our present knowledge of the primary structures of β subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with α1 subunits, the effects of β subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by β subunits, such as long-lasting prepulse facilitation of α1C (absent in α1E) and inhibition by G protein βγ dimer of α1E, absent in α1C. One β subunit, a brain β2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of α1 in oocytes requires a β subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca2+ channels to account for “α1 alone” channels seen in cells with limited β subunit expression. In one model, β dissociates from the mature α1 after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of β would then have subunit composition α1β. In the other model, the “chaperoning” β remains associated with the mature channel and “α1 alone” channels would in fact be α1β channels. Upon co-expression of high levels of β the regulated channels would have composition [α1β]β.

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.

Early afterdepolarizations in cardiac myocytes: beyond reduced repolarization reserve

Early afterdepolarizations (EADs) are secondary voltage depolarizations during the repolarizing phase of the action potential, which can cause lethal cardiac arrhythmias. The occurrence of EADs requires a reduction in outward current and/or an increase in inward current, a condition called reduced repolarization reserve. However, this generalized condition is not sufficient for EAD genesis and does not explain the voltage oscillations manifesting as EADs. Here, we summarize recent progress that uses dynamical theory to build on and advance our understanding of EADs beyond the concept of repolarization reserve, towards the goal of developing a holistic and integrative view of EADs and their role in arrhythmogenesis. We first introduce concepts from nonlinear dynamics that are relevant to EADs, namely, Hopf bifurcation leading to oscillations and basin of attraction of an equilibrium or oscillatory state. We then present a theory of phase-2 EADs in nonlinear dynamics, which includes the formation of quasi-equilibrium states at the plateau voltage, their stabilities, and the bifurcations leading to and terminating the oscillations. This theory shows that the L-type calcium channel plays a unique role in causing the nonlinear dynamical behaviours necessary for EADs. We also summarize different mechanisms of phase-3 EADs. Based on the dynamical theory, we discuss the roles of each of the major ionic currents in the genesis of EADs, and potential therapeutic targets.

Hormonal control of protein expression and mRNA levels of the MaxiK channel α subunit in myometrium

Large conductance voltage‐dependent and Ca2+‐modulated K+ channels play a crucial role in myometrium contractility. Western blots and immunocytochemistry of rat uterine sections or isolated cells show that MaxiK channel protein signals drastically decrease towards the end of pregnancy. Consistent with a transcriptional regulation of channel expression, mRNA levels quantified with the ribonuclease protection assay correlated well with MaxiK protein levels. As a control, Na+/K+‐ATPase protein and RNA levels do not significantly change at different stages of pregnancy. The low numbers of MaxiK channels at the end of pregnancy may facilitate uterine contraction needed for parturition.

Coupling between charge movement and pore opening in vertebrate neuronal alpha 1E calcium channels.

1. Neuronal alpha 1E Ca2+ channels were expressed alone and in combination with the beta 2a subunit in Xenopus laevis oocytes. 2. The properties of ionic and gating currents of alpha 1E were investigated: ionic currents were measured in 10 mM external Ba2+; gating currents were isolated in 2 mM external Co2+. 3. Charge movement preceded channel opening. The charge movement voltage curve (Q(V)) preceded the ionic conductance voltage dependence (G(V)) by approximately 20 mV. 4. Coexpression of alpha 1E with the beta 2a subunit did not modify the voltage dependence of charge movement but shifted the G(V) curve to more negative potentials. The voltage gap between Q(V) and G(V) curves was reduced by the beta 2a subunit and both curves overlapped at potentials near 0 mV. 5. The coupling efficiency between the charge movement and pore opening was estimated by the ration between limiting conductance and maximum charge movement (Gmax/Qmax). Coexpression of the beta 2a subunit increased the Gmax/Qmax ratio from 9.2 x 10(5) +/‐ 1.4 x 10(5) to 21.9 x 10(5) +/‐ 2.8 X 10(5) S C‐1 for alpha 1E and alpha 1E + beta 2a, respectively. 6. We conclude that in the neuronal alpha 1E the charge movement is tightly coupled with the pore opening and that the beta 2a subunit coexpression further improves this coupling.

Transduction of voltage and Ca2+ signals by Slo1 BK channels.

Large-conductance Ca2+ -and voltage-gated K+ channels are activated by an increase in intracellular Ca2+ concentration and/or depolarization. The channel activation mechanism is well described by an allosteric model encompassing the gate, voltage sensors, and Ca2+ sensors, and the model is an excellent framework to understand the influences of auxiliary β and γ subunits and regulatory factors such as Mg2+. Recent advances permit elucidation of structural correlates of the biophysical mechanism.

A Single M1 Residue in the β2 Subunit Alters Channel Gating of GABAA Receptor in Anesthetic Modulation and Direct Activation

General anesthetics allosterically modulate the activity of neuronal γ-aminobutyric acid, type A (GABAA), receptors. Previous mutational studies from our laboratory and others have shown that the regions in transmembrane domain 1 (M1) and pre-M1 of α and β subunits in GABA receptors are essential for positive modulation of GABA binding and function by the intravenous (IV) general anesthetics. Mutation of β2Gly-219 to Phe corresponded in ρ nearly eliminated the modulatory effect of IV anesthetics in α1/β2/γ2S combination. However, the general anesthetics retained the ability to directly open the channel of mutant G219F, and the apparent affinity for GABA was increased, and desensitization rate was reduced. In this study, we made additional single mutations such as 219 Ser, Cys, Ile, Asp, Arg, Tyr, and Trp. The larger side chains of the replacement residues produced the greatest reduction in enhancement of GABA currents by IV anesthetics at clinical concentrations (Trp > Tyr = Phe > Arg > Asp > Ile > Cys > Ser = wild type). Compared with a 2–3-fold response in wild type, pentobarbital and propofol enhanced less than 0.5-fold; etomidate and alphaxalone modulation was reduced from more than 4- to 1-fold in G219F, G219Y, and G219W. A linear correlation was observed between the volume of the residue at position 219 and the loss of modulation. An identical correlation was found for the effect of modulation on left-shift in the GABA EC50 value; furthermore, the same rank order of residues, related to size, was found for reduction in the maximal direct channel-gating by pentobarbital (1 mm) and etomidate (100 μm) and for increased apparent affinity for direct gating by the IV anesthetics.

Voltage-dependent conformational changes in human Ca2+- and voltage-activated K+ channel, revealed by voltage-clamp fluorometry

Large conductance voltage- and Ca2+-activated K+ (BKCa) channels regulate important physiological processes such as neurotransmitter release and vascular tone. BKCa channels possess a voltage sensor mainly represented by the S4 transmembrane domain. Changes in membrane potential displace the voltage sensor, producing a conformational change that leads to channel opening. By site-directed fluorescent labeling of residues in the S3–S4 region and by using voltage clamp fluorometry, we have resolved the conformational changes the channel undergoes during activation. The voltage dependence of these conformational changes (detected as changes in fluorescence emission, fluorescence vs. voltage curves) always preceded the channel activation curves, as expected for protein rearrangements associated to the movement of the voltage sensor. Extremely slow conformational changes were revealed by fluorescent labeling of position 202, elicited by a mutual interaction of the fluorophore with the adjacent tryptophan 203.

Regulation of K+ flow by a ring of negative charges in the outer pore of BKCa channels. Part I: Aspartate 292 modulates K+ conduction by external surface charge effect.

The pore region of the majority of K+ channels contains the highly conserved GYGD sequence, known as the K+ channel signature sequence, where the GYG is critical for K+ selectivity (Heginbotham, L., T. Abramson, and R. MacKinnon. 1992. Science. 258:1152–1155). Exchanging the aspartate residue with asparagine in this sequence abolishes ionic conductance of the Shaker K+ channel (D447N) (Hurst, R.S., L. Toro, and E. Stefani. 1996. FEBS Lett. 388:59–65). In contrast, we found that the corresponding mutation (D292N) in the pore forming α subunit (hSlo) of the voltage- and Ca2+-activated K+ channel (BKCa, MaxiK) did not prevent conduction but reduced single channel conductance. We have investigated the role of outer pore negative charges in ion conduction (this paper) and channel gating (Haug, T., R. Olcese, T. Ligia, and E. Stefani. 2004. J. Gen Physiol. 124:185–197). In symmetrical 120 mM [K+], the D292N mutation reduced the outward single channel conductance by ∼40% and nearly abolished inward K+ flow (outward rectification). This rectification was partially relieved by increasing the external K+ concentration to 700 mM. Small inward currents were resolved by introducing an additional mutation (R207Q) that greatly increases the open probability of the channel. A four-state multi-ion pore model that incorporates the effects of surface charge was used to simulate the essential properties of channel conduction. The conduction properties of the mutant channel (D292N) could be predicted by a simple ∼8.5-fold reduction of the surface charge density without altering any other parameter. These results indicate that the aspartate residue in the BKCa pore plays a key role in conduction and suggest that the pore structure is not affected by the mutation. We speculate that the negative charge strongly accumulates K+ in the outer vestibule close to the selectivity filter, thus increasing the rate of ion entry into the pore.

Molecular Coupling between Voltage Sensor and Pore Opening in the Arabidopsis Inward Rectifier K+ Channel KAT1

Animal and plant voltage-gated ion channels share a common architecture. They are made up of four subunits and the positive charges on helical S4 segments of the protein in animal K+ channels are the main voltage-sensing elements. The KAT1 channel cloned from Arabidopsis thaliana, despite its structural similarity to animal outward rectifier K+ channels is, however, an inward rectifier. Here we detected KAT1-gating currents due to the existence of an intrinsic voltage sensor in this channel. The measured gating currents evoked in response to hyperpolarizing voltage steps consist of a very fast (τ = 318 ± 34 μs at −180 mV) and a slower component (4.5 ± 0.5 ms at −180 mV) representing charge moved when most channels are closed. The observed gating currents precede in time the ionic currents and they are measurable at voltages (less than or equal to −60) at which the channel open probability is negligible (≈10−4). These two observations, together with the fact that there is a delay in the onset of the ionic currents, indicate that gating charge transits between several closed states before the KAT1 channel opens. To gain insight into the molecular mechanisms that give rise to the gating currents and lead to channel opening, we probed external accessibility of S4 domain residues to methanethiosulfonate-ethyltrimethylammonium (MTSET) in both closed and open cysteine-substituted KAT1 channels. The results demonstrate that the putative voltage–sensing charges of S4 move inward when the KAT1 channels open.