Claudia M. Moreno

Dystrophin-deficient cardiomyocytes derived from human urine: New biologic reagents for drug discovery

The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patients skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patients dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.

Graded Ca2+/calmodulin-dependent coupling of voltage-gated CaV1.2 channels

In the heart, reliable activation of Ca2+ release from the sarcoplasmic reticulum during the plateau of the ventricular action potential requires synchronous opening of multiple CaV1.2 channels. Yet the mechanisms that coordinate this simultaneous opening during every heartbeat are unclear. Here, we demonstrate that CaV1.2 channels form clusters that undergo dynamic, reciprocal, allosteric interactions. This ‘functional coupling’ facilitates Ca2+ influx by increasing activation of adjoined channels and occurs through C-terminal-to-C-terminal interactions. These interactions are initiated by binding of incoming Ca2+ to calmodulin (CaM) and proceed through Ca2+/CaM binding to the CaV1.2 pre-IQ domain. Coupling fades as [Ca2+]i decreases, but persists longer than the current that evoked it, providing evidence for ‘molecular memory’. Our findings suggest a model for CaV1.2 channel gating and Ca2+-influx amplification that unifies diverse observations about Ca2+ signaling in the heart, and challenges the long-held view that voltage-gated channels open and close independently. DOI: http://dx.doi.org/10.7554/eLife.05608.001

STIM1 and Orai1 mediate thrombin-induced Ca2+ influx in rat cortical astrocytes

In astrocytes, thrombin leads to cytoplasmic Ca(2+) elevations modulating a variety of cytoprotective and cytotoxic responses. Astrocytes respond to thrombin stimulation with a biphasic Ca(2+) increase generated by an interplay between ER-Ca(2+) release and store-operated Ca(2+) entry (SOCE). In many cell types, STIM1 and Orai1 have been demonstrated to be central components of SOCE. STIM1 senses the ER-Ca(2+) depletion and binds Orai1 to activate Ca(2+) influx. Here we used immunocytochemistry, overexpression and siRNA assays to investigate the role of STIM1 and Orai1 in the thrombin-induced Ca(2+) response in primary cultures of rat cortical astrocytes. We found that STIM1 and Orai1 are endogenously expressed in cortical astrocytes and distribute accordingly with other mammalian cells. Importantly, native and overexpressed STIM1 reorganized in puncta under thrombin stimulation and this reorganization was reversible. In addition, the overexpression of STIM1 and Orai1 increased by twofold the Ca(2+) influx evoked by thrombin, while knockdown of endogenous STIM1 and Orai1 significantly decreased this Ca(2+) influx. These results indicate that STIM1 and Orai1 underlie an important fraction of the Ca(2+) response that astrocytes exhibit in the presence of thrombin. Thrombin stimulation in astrocytes leads to ER-Ca(2+) release which causes STIM1 reorganization allowing the activation of Orai1 and the subsequent Ca(2+) influx.

Combined single channel and single molecule detection identifies subunit composition of STIM1-activated transient receptor potential canonical (TRPC) channels.

Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity.

Ca2+ entry into neurons is facilitated by cooperative gating of clustered CaV1.3 channels

CaV1.3 channels regulate excitability in many neurons. As is the case for all voltage-gated channels, it is widely assumed that individual CaV1.3 channels behave independently with respect to voltage-activation, open probability, and facilitation. Here, we report the results of super-resolution imaging, optogenetic, and electrophysiological measurements that refute this long-held view. We found that the short channel isoform (CaV1.3S), but not the long (CaV1.3L), associates in functional clusters of two or more channels that open cooperatively, facilitating Ca2+ influx. CaV1.3S channels are coupled via a C-terminus-to-C-terminus interaction that requires binding of the incoming Ca2+ to calmodulin (CaM) and subsequent binding of CaM to the pre-IQ domain of the channels. Physically-coupled channels facilitate Ca2+ currents as a consequence of their higher open probabilities, leading to increased firing rates in rat hippocampal neurons. We propose that cooperative gating of CaV1.3S channels represents a mechanism for the regulation of Ca2+ signaling and electrical activity. DOI: http://dx.doi.org/10.7554/eLife.15744.001

SOC and now also SIC: Store‐operated and store‐inhibited channels

There is a specialized form of calcium influx that involves a close communication between endoplasmic reticulum and the channels at the plasma membrane. In one side store depletion activates channels known as store‐operated channels (SOC), which are responsible of the well‐studied store‐operated calcium entry (SOCE). SOC comprises two different types of channels. Orai, which is exclusively activated by store depletion being the channel responsible of the calcium release‐activated calcium current, and transient receptor potential canonical channel, which in contrast, is activated by store depletion only under specific conditions and carries nonselective cationic currents. On the other hand, it has been recently shown that store depletion also inhibits calcium channels. The first member identified, of what we named as store‐inhibited channels (SIC), is the L‐type voltage‐gated calcium channel. Stores control both SOC and SIC by means of the multifunctional protein STIM1. The identification of SOC and SIC opens a new scenario for the role of store depletion in the modulation of different calcium entry pathways, which may satisfy different cellular processes.© 2011 IUBMB IUBMB Life, 2011.

Distance constraints on activation of TRPV4 channels by AKAP150-bound PKCα in arterial myocytes

TRPV4 (transient receptor potential vanilloid 4) channels are Ca2+-permeable channels that play a key role in regulating vascular tone. In arterial myocytes, opening of TRPV4 channels creates local increases in Ca2+ influx, detectable optically as “TRPV4 sparklets.” TRPV4 sparklet activity can be enhanced by the action of the vasoconstrictor angiotensin II (AngII). This modulation depends on the activation of subcellular signaling domains that comprise protein kinase C &agr; (PKC&agr;) bound to the anchoring protein AKAP150. Here, we used super-resolution nanoscopy, patch-clamp electrophysiology, Ca2+ imaging, and mathematical modeling approaches to test the hypothesis that AKAP150-dependent modulation of TRPV4 channels is critically dependent on the distance between these two proteins in the sarcolemma of arterial myocytes. Our data show that the distance between AKAP150 and TRPV4 channel clusters varies with sex and arterial bed. Consistent with our hypothesis, we further find that basal and AngII-induced TRPV4 channel activity decays exponentially as the distance between TRPV4 and AKAP150 increases. Our data suggest a maximum radius of action of ∼200 nm for local modulation of TRPV4 channels by AKAP150-associated PKC&agr;.

Proximal clustering between BK and CaV1.3 channels promotes functional coupling and BK channel activation at low voltage

CaV-channel dependent activation of BK channels is critical for feedback control of both calcium influx and cell excitability. Here we addressed the functional and spatial interaction between BK and CaV1.3 channels, unique CaV1 channels that activate at low voltages. We found that when BK and CaV1.3 channels were co-expressed in the same cell, BK channels started activating near −50 mV, ~30 mV more negative than for activation of co-expressed BK and high-voltage activated CaV2.2 channels. In addition, single-molecule localization microscopy revealed striking clusters of CaV1.3 channels surrounding clusters of BK channels and forming a multi-channel complex both in a heterologous system and in rat hippocampal and sympathetic neurons. We propose that this spatial arrangement allows tight tracking between local BK channel activation and the gating of CaV1.3 channels at quite negative membrane potentials, facilitating the regulation of neuronal excitability at voltages close to the threshold to fire action potentials. DOI: http://dx.doi.org/10.7554/eLife.28029.001

Microdomain Organization and the Role of Second Messengers

Store-operated calcium entry (SOCE) occurs at specialized regions where the endoplasmic reticulum and plasma membranes are closely apposed. Several molecules converge in these junctions to form a complex that spatiotemporally circumscribes SOCE signaling. We have named recently this complex as SOCIC (Store Operated Calcium Influx Complex). There is a growing list of SOCIC members, including the Ca2+ sensor and channel activator STIM1, the Orai and TRPC1 channels, SOCE regulators as CaM and CRACR2A, and SOCE-regulated proteins as SERCA and adenylyl cyclases. Considering that under physiological conditions Ca2+ entry is transient, SOCIC should be a dynamic structure that goes through assembly and disassembly cycles depending on cell requirements, and on the depleted state of intracellular Ca2+ stores. Moreover SOCIC seems to assembly at specialized regions of plasma membrane known as lipid rafts. In this chapter we discuss the evidence supporting the idea that SOCE occurs at microdomains and introduce the SOCIC components known so far. Then we illustrate some ideas on how this complex is assembled and disassembled. Finally we address the evidence of physiological and pathological implications of the microdomain organization of SOCE.

BIN1 induces the formation of T-tubules and adult-like Ca2+ release units in developing cardiomyocytes: BIN1 promotes hESC-CMs with ventricular phenotype

Human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs) are at the center of new cell‐based therapies for cardiac disease, but may also serve as a useful in vitro model for cardiac cell development. An intriguing feature of hESC‐CMs is that although they express contractile proteins and have sarcomeres, they do not develop transverse‐tubules (T‐tubules) with adult‐like Ca2+ release units (CRUs). We tested the hypothesis that expression of the protein BIN1 in hESC‐CMs promotes T‐tubules formation, facilitates CaV1.2 channel clustering along the tubules, and results in the development of stable CRUs. Using electrophysiology, [Ca2+]i imaging, and super resolution microscopy, we found that BIN1 expression induced T‐tubule development in hESC‐CMs, while increasing differentiation toward a more ventricular‐like phenotype. Voltage‐gated CaV1.2 channels clustered along the surface sarcolemma and T‐tubules of hESC‐CM. The length and width of the T‐tubules as well as the expression and size of CaV1.2 clusters grew, as BIN1 expression increased and cells matured. BIN1 expression increased CaV1.2 channel activity and the probability of coupled gating within channel clusters. Interestingly, BIN1 clusters also served as sites for sarcoplasmic reticulum (SR) anchoring and stabilization. Accordingly, BIN1‐expressing cells had more CaV1.2‐ryanodine receptor junctions than control cells. This was associated with larger [Ca2+]i transients during excitation–contraction coupling. Our data support the view that BIN1 is a key regulator of T‐tubule formation and CaV1.2 channel delivery. By studying the role of BIN1 during the differentiation of hESC‐CMs, we show that BIN1 is also important for CaV1.2 channel clustering, junctional SR organization, and the establishment of excitation–contraction coupling. Stem Cells 2019;37:54–64