Papers by Riccardo Olcese
The Journal of General Physiology, Jul 16, 2012
Large-conductance voltage-and Ca-activated K + (BK) channels are potent regulators of excitabilit... more Large-conductance voltage-and Ca-activated K + (BK) channels are potent regulators of excitability, broadly expressed in most mammalian cells (Toro et al., 1998; Latorre and Brauchi, 2006; Salkoff et al., 2006). BK channels are formed by -subunit homotetramers (Shen et al., 1994) (Fig. 1), each comprising a conserved transmembrane voltage-sensing domain (VSD), helices contributing to the ion-conducting pore domain, and large intracellular ligand-binding domains that assemble into the gating ring superstructure in the tetramer (Wang
The Journal of General Physiology, Nov 1, 1997
Biophysical Journal, Feb 1, 2020
Mitochondria are dynamic organelles that constantly undergo fission and fusion. A key player in m... more Mitochondria are dynamic organelles that constantly undergo fission and fusion. A key player in mitochondrial fission, dynamin-related protein 1 (Drp1), is recruited from the cytosol to pre-destined fission sites at the mitochondrial outer membrane by membrane-anchored protein adaptors, including mitochondrial dynamics protein of 51 kDa (MiD51). The nucleotidyl transferase domain of MiD51 does not exhibit enzymatic activity, yet selectively binds ADP. The role of ADP in MiD51 function is unclear. We have used microscale thermophoresis (MST) and X-ray crystallography to reveal the direct, selective interaction of MiD51 with cone-shaped phospholipids that induce negative curvature stress to achieve local membrane constriction. We found that MiD51 specifically binds cardiolipin (CL) and phosphatidic acid (PA), both of which play critical roles in the regulation of Drp1-mediated membrane fission. Interestingly, ADP competitively inhibits MiD51-CL binding suggesting that CL and ADP, both of which possess dianionic phosphates at physiological pH, bind overlapping binding sites in MiD51. We corroborated this result using a nucleotide-binding disruption mutant of MiD51, which also showed reduced CL binding affinity. Further, the absence of interactions with shorter acyl chain 6-phosphatidic acid (6-PA) indicated that longer acyl chains (or greater hydrophobic surface area) are required for efficient MiD51-phospholipid interactions. The crystal structure of PA-bound MiD51 shows that PA binding transforms the disordered N-terminal region into an a-helix. Docking studies reveal that CL has a similar effect. Overall, our results suggest that ADP negatively regulates MiD51-CL interactions, which likely functions to couple the energetic state of the cell (i.e. ATP/ADP ratio) to the balance of mitochondrial fission and fusion.
Biophysical Journal, Feb 1, 2016
channels have been shown to be localized to the intercalated disks along with Cx43 channels. Rece... more channels have been shown to be localized to the intercalated disks along with Cx43 channels. Recent evidence of reciprocity in the co-localized expression of Kir2.1 and Nav1.5 channels in cardiac myocytes suggest that ionic currents due to these two channels are in some way calibrated to each other. In isolated cells, the fast sodium current is much larger than IK1 resulting in a large depolarization reserve. Using simulations of chains of cardiac cells, we show that the depolarization reserve for conducting action potentials is significantly smaller than in isolated cells. We also studied the changes in the depolarization reserve with variations in gap junction channel density and explored the conditions under which propagation slowed or failed. These insights will allow a better understanding of the effects of Na channel blockers as well as regional differences in action potential conduction in the heart.
International Review of Neurobiology, 2016
BK channels are universal regulators of cell excitability, given their exceptional unitary conduc... more BK channels are universal regulators of cell excitability, given their exceptional unitary conductance selective for K(+), joint activation mechanism by membrane depolarization and intracellular [Ca(2+)] elevation, and broad expression pattern. In this chapter, we discuss the structural basis and operational principles of their activation, or gating, by membrane potential and calcium. We also discuss how the two activation mechanisms interact to culminate in channel opening. As members of the voltage-gated potassium channel superfamily, BK channels are discussed in the context of archetypal family members, in terms of similarities that help us understand their function, but also seminal structural and biophysical differences that confer unique functional properties.
The Journal of Physiology, Jul 11, 2019
Key points Association of plasma membrane BKCa channels with BK‐β subunits shapes their biophysic... more Key points Association of plasma membrane BKCa channels with BK‐β subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BKCa channel (mitoBKCa) by BK‐β subunits is not established. MitoBKCa‐α and the regulatory BK‐β1 subunit associate in mouse cardiac mitochondria. A large fraction of mitoBKCa display properties similar to that of plasma membrane BKCa when associated with BK‐β1 (left‐shifted voltage dependence of activation, V1/2 = −55 mV, 12 µm matrix Ca2+). In BK‐β1 knockout mice, cardiac mitoBKCa displayed a low Po and a depolarized V1/2 of activation (+47 mV at 12 µm matrix Ca2+) Co‐expression of BKCa with the BK‐β1 subunit in HeLa cells doubled the density of BKCa in mitochondria. The present study supports the view that the cardiac mitoBKCa channel is functionally modulated by the BK‐β1 subunit; proper targeting and activation of mitoBKCa shapes mitochondrial Ca2+ handling. Association of the plasma membrane BKCa channel with auxiliary BK‐β1–4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBKCa) with regulatory subunits is unknown. We report that mitoBKCa functionally associates with its regulatory subunit BK‐β1 in adult rodent cardiomyocytes. Cardiac mitoBKCa is a calcium‐ and voltage‐activated channel that is sensitive to paxilline with a large conductance for K+ of 300 pS. Additionally, mitoBKCa displays a high open probability (Po) and voltage half‐activation (V1/2 = −55 mV, n = 7) resembling that of plasma membrane BKCa when associated with its regulatory BK‐β1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BKCa‐α and its BK‐β1 subunit. Mitochondria from the BK‐β1 knockout (KO) mice showed sparse mitoBKCa currents (five patches with mitoBKCa activity out of 28 total patches from n = 5 different hearts), displaying a depolarized V1/2 of activation (+47 mV in 12 µm matrix Ca2+). The reduced activity of mitoBKCa was accompanied by a high expression of BKCa transcript in the BK‐β1 KO, suggesting a lower abundance of mitoBKCa channels in this genotype. Accordingly, BK‐β1subunit increased the localization of BKDEC (i.e. the splice variant of BKCa that specifically targets mitochondria) into mitochondria by two‐fold. Importantly, both paxilline‐treated and BK‐β1 KO mitochondria displayed a more rapid Ca2+ overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBKCa associates with its regulatory BK‐β1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBKCa channel that helps to maintain mitochondrial Ca2+ homeostasis.
Biophysical Journal, Feb 1, 2021
Transgenic mice expressing high levels of human SOD1 G93A (mutant SOD1 or mSOD1) 1 and their wild... more Transgenic mice expressing high levels of human SOD1 G93A (mutant SOD1 or mSOD1) 1 and their wild-type (WT) littermates were used for all the experiments (JAX Strain: 002726 1 B6SJL-Tg (SOD1*G93A)1Gur/J). All animal protocols were approved by the Institutional 1 Animal Care and Use Committee at UCLA. Experiments were performed at postnatal week 2 (8 1-14 day old mice of either gender) when the rhythmic jaw movements and suckling behavior are 1 fully developed (Turman, 2007). Genotype of mice was determined by standard PCR technique 1 using tails samples (Laragen, Inc, CA). Experimental preparations and techniques include: 1) 1 live brainstem slices to conduct in vitro whole-cell current-clamp, voltage-clamp and dynamic-1 clamp electrophysiology from Mes V sensory neurons, 2) acutely dissociated trigeminal ganglion 1 neurons to conduct current-clamp experiments, 3) live whole retinal preparation to conduct 1 current-clamp experiments, 4) fixed cryosectioned coronal pontine sections for Nav1.6 protein 1 quantification, and, 5) computational model of Mes V-TMN network to investigate a functional 1 consequence of sensory abnormality on motor discharge. I. In vitro patch-clamp electrophysiology 1 a. Brainstem slice preparation for Mes V electrophysiology Brain slices were prepared and used for conducting whole-cell current-, voltage-and dynamic-clamp electrophysiology in the brainstem primary sensory neurons of the trigeminal 1 Mes V nucleus. Pups were anesthetized using isoflurane vapor inhalation, and decapitated. The 1 head was immediately immersed in carboxygenated (95% O 2-5% CO 2), ice-cold sucrose cutting 1
Biophysical Journal, Feb 1, 2019
targeted therapies. As many of these mutations have been shown to impair channel inactivation, ca... more targeted therapies. As many of these mutations have been shown to impair channel inactivation, calcium channel blockers (CCBs) such as verapamil and dihydropyridines (DHPs) represent a promising option for treatment, however, these CCBs often prove only partially effective for managing symptoms in these patients. We propose that this lack of efficacy is caused by attenuated channel inactivation in the mutant channels. In particular, verapamil and DHPs are known to preferentially block open and inactivated channels such that loss of entry into the inactivated state would decrease the efficacy of these CCBs on the mutant channels. Here, we demonstrate that mutations in Ca V 1.2 which diminish channel inactivation also decrease the extent of channel block by CCBs. Furthermore, we show that amount of channel block by these CCBs is strongly correlated to the fraction of inactivation in the channel, whereby efficacy of block is lowest in mutant channels with the lowest fraction of inactivation. This decreased efficacy of CCBs on mutant channels represents a major impediment to the treatment of TS and cardiac arrhythmias caused by mutations in Ca V 1.2. These results emphasize the need to tailor patient therapies to the specific channel deficit produced by each mutation.
Biophysical Journal, Feb 1, 2019
Recent studies report that a naturally-occurring mutation in the a 1S subunit of voltage-gated, s... more Recent studies report that a naturally-occurring mutation in the a 1S subunit of voltage-gated, skeletal Ca 2þ channels Ca V 1.1 is linked to malignant hyperthermia and prevents Ca 2þ influx in myotubes, leaving excitationcontraction coupling largely unaffected (Eltit et al., 2012, PNAS; Bannister & Beam, 2013, Biophys J). The mutation is located in the voltage sensing domain (VSD) of Repeat I (IS4) and neutralizes its innermost basic amino acid (R174W). To gain a mechanistic understanding of how this mutation alters channel function, we optically tracked the activation of VSDI of human Ca V 1.1 channels using the voltage-clamp fluorometry technique. We expressed Ca V 1.1 channel complexes formed by a 1s (WT or R174W), STAC3 and b 1a subunits in Xenopus oocytes and labeled VSD I with thiol-reactive fluorophores at a strategically-introduced Cysteine (L159C) located at the extracellular flank of IS4. The mutant channel exhibited a right-shifted voltage dependence of channel activation by 60mV, explaining the lack of Ca 2þ influx at physiological membrane potentials in myotubes experiments (Eltit et al., 2012, PNAS). Simultaneous to ionic current recordings, we resolved voltage-dependent fluorescence changes from VSD I, which report the movement of this voltage sensor. We found that the malignant-hyperthermia-linked mutation compromised VSD I voltage dependent activation, causing a right shift (40mV) of the F(V) curve and a decrease of its effective valence by 50%. That is, the R174W mutation stabilizes the resting state of VSD I, thus impairing channel opening. This finding suggests a contribution of VSD I in channel activation in CaV1.1 channels in this specific subunit composition.
Biophysical Journal, Feb 1, 2019
Using the cut-open oocyte voltage clamp technique, we recorded ionic currents from channel comple... more Using the cut-open oocyte voltage clamp technique, we recorded ionic currents from channel complexes formed by a 1S /b 1a /stac3 with or without g 1 subunit, expressedin Xenopus oocytes. g 1 modified Ca V 1.1 voltage dependence of inactivation by decreasing the effective valence from 5.351.1e 0 (N=4) to 1.8750.2e 0 (N=4) and reducing the non-inactivating component of the ionic current following 10s pulses from $50% to $30%. In addition, we observed that g 1 coexpression did not affect voltage dependence of Ca V 1.1 activation (No g 1 : V half = 37.151.2mV, N=5, and with g 1 : V half = 40.051.5mV, N=4). However, when g 1 and a 2 d-1 were coexpressed, the voltage dependence of the Ca V 1.1 complex (a 1S /b 1a /a 2 d-1/g 1 /stac3) was facilitated. g 1 also strongly modulated the Ca V 1.2 channel isoform (a 1C / b 2b /a 2 d-1) by shifting the voltage dependence of channel inactivation by $20mV toward negative potentials. In conclusion, these studies have revealed the biophysical consequences of g 1 subunit association with two distinct human L-type channel isoforms in heterologous expression systems.
Biophysical Journal, Feb 1, 2018
form pathways for lipophilic drugs that modulate slow-inactivation; hence, our findings provide n... more form pathways for lipophilic drugs that modulate slow-inactivation; hence, our findings provide novel insights into the molecular basis for state-dependent effects of these drugs on channel function.
Biophysical Journal, Feb 1, 2017
Biophysical Journal, Feb 1, 2016
In vitro manipulation of membrane sterol level has an impact on the regulation of ion channels, h... more In vitro manipulation of membrane sterol level has an impact on the regulation of ion channels, however, a comprehensive study is lacking to confirm the physiological or pathophysiological significance of these experiment. Smith-Lemli-Opitz syndrome (SLOS) is characterized by a reduced or abolished activity of 7-dehydrocholesterol (7DHC) reductase, which leads to the elevation of the 7DHC in the tissues and blood. We utilized T cells isolated from SLOS patients to address the question if in vivo altered membrane sterol composition impairs the operation of Kv1.3, the predominant voltage-gated ion channel of T cells, and if altered Kv1.3 function is reflected in impaired mitogenic responses of SLOS T lymphocytes. Using whole-cell patch-clamp technique we showed that the activation kinetics of Kv1.3 is slower and the midpoint of the voltage-dependence of steady-state activation is shifted to depolarized potentials in SLOS T cells as compared to age-matched controls. Similar changes in the kinetic and equilibrium parameters of Kv1.3 gating were detected in control T cells loaded with 7DHC using 7DHC/cyclodextrin complex. Upon removal of putative sterol binding sites of Kv1.3 the channel become insensitive to 7DHC loading. Functional assays revealed that modified operation of Kv1.3 in the SLOS T cells is associated with impaired proliferation rate (CFSE method) and a defect in the early steps of Kv1.3:-and Ca 2þ-dependent activation process (CD154/CD40L expression) in CD3 þ cells. Our conclusion is that the function of Kv1.3 is modified in SLOS via a direct coupling to the 7DHC in the cell membrane. We propose that this ion channel-sterol interaction reveals a molecular mechanism that may contribute to the pathophysiological conditions in SLOS and lead to the most prominent neurological and cardiovascular symptoms via influencing the physiological function of ion channels.
Large-conductance Ca 2ϩ-and voltage-gated K ϩ channels are activated by an increase in intracellu... more Large-conductance Ca 2ϩ-and voltage-gated K ϩ channels are activated by an increase in intracellular Ca 2ϩ concentration and/or depolarization. The channel activation mechanism is well described by an allosteric model encompassing the gate, voltage sensors, and Ca 2ϩ sensors, and the model is an excellent framework to understand the influences of auxiliary  and ␥ subunits and regulatory factors such as Mg 2ϩ. Recent advances permit elucidation of structural correlates of the biophysical mechanism.
Biophysical Journal, 2014
this increase in the Ca 2þ spark rate was associated with a decrease in the SR Ca 2þ content. Thi... more this increase in the Ca 2þ spark rate was associated with a decrease in the SR Ca 2þ content. This is consistent with the increase in the SR Ca 2þ leak (as evidenced by the increase in the Ca 2þ spark rate) that followed the H 2 O 2 application. Since ROS has been shown to activate other signaling systems in heart (e.g. CaMKII), the interactions between H 2 O 2 dependent ROS elevation and both CaMKII and PKA were examined. While significant interactions between rapid, transient ROS elevation and CaMKII and PKA were observed, it was also determined that the actions of these ROS elevations on Ca 2þ sparks was not mediated by either CaMKII or PKA. How ROS may affect EC coupling under these conditions is also examined and discussed.
Biophysical Journal, 2013
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Papers by Riccardo Olcese