Channels In Excitable Cells Research Articles

Degradation of paralytic shellfish toxins during flocculation of Alexandrium pacificum by an oxidized modified clay: A laboratory experiment

Paralytic shellfish toxins (PSTs), produced by Alexandrium pacificum in the marine environment, are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells. During the toxigenic A. pacificum blooms outbreaks, PSTs can be accumulated through the food chain and finally enter the human body, posing a significant threat to human health and safety. This study experimented with a novel type of oxidized modified clay, potassium peroxymonosulfate modified clay (PMPS-MC), which could remove A. pacificum cells as well as reduce intracellular and extracellular PSTs toxicity rapidly. For the extracellular PSTs, its content decreased to below the detection limit rapidly through oxidative degradation within 15 min of 10 mg/L PMPS-MC treatment. Whereafter, although the residual cells in water column and some viable cells in flocculated sediment continued to secrete toxins, the extracellular PSTs content and toxicity in the PMPS-MC treatment groups remained significantly lower than those in the control group. For the intracellular PSTs, PMPS-MC might induce the transformation of more toxic GTX1&4 to less toxic GTX2&3 and C1&2, resulting in intracellular PSTs toxicity reduced within 15 min. In addition, intracellular PSTs content and toxicity in the PMPS-MC treatment groups were consistently lower than the control group within 48 h, possibly by inhibiting the A. pacificum cells growth. These results will provide a scientific basis for the field application of modified clay to control A. pacificum blooms.

Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes

Mammalian voltage-activated L-type Ca2+ channels, such as Ca(v)1.2, control transmembrane Ca2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N terminus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current-voltage relationship when α1C was not palmitoylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but significantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca2+ channels in excitable cells.

Tetralol derivative NNC-55-0396 induces glioblastoma cell death by activating IRE1α, JNK1 and calcium signaling

Mibefradil and NNC-55–0396, tetralol derivatives with a proven –ability to block T-type calcium channels in excitable cells, reduce cancer cell viability in vitro, causing cell death. Furthermore, they reduce tumor growth in preclinical models of Glioblastoma multiforme (GBM), a brain tumor of poor prognosis. Here we found that GBM cells treated with cytotoxic concentrations of NNC-55–0396 paradoxically increased cytosolic calcium levels through the activation of inositol triphosphate receptors (IP3R) and ER stress. We used pharmacological inhibitors and gene silencing to dissect the cell death pathway stimulated by NNC-55-0396 in GBM cell lines and biopsy-derived cultures. Calcium chelation or IP3R inhibition prevented NNC-55–0396-mediated cytotoxicity, indicating that ER calcium efflux is the cause of cell death. Upstream of calcium mobilization, NNC-55–0396 activated the IRE1α arm of the Unfolded Protein Response (UPR) resulting in the nuclear translocation of pro-apoptotic CHOP. Consistent with these findings, silencing IRE1α or JNK1 rescued the cell death elicited by NNC-55–0396. Therefore, we demonstrate that activation of IRE1α and calcium signaling accounts for the cytotoxicity of NNC-55–0396 in GBM cells. The delineation of the signaling pathway that mediates the abrupt cell death triggered by this compound can help the development of new therapies for GBM.

High-Throughput Analysis of Drug Safety Responses in Induced Pluripotent Stem Cell-Derived Cardiomyocytes Using Multielectrode Array.

Microelectrode array (MEA) isan electrophysiological instrument used to track activities of ion channels in excitable cells. Neurons and cardiomyocytes are seeded to form a cell monolayer on a field of sensors able to detect electrical signals, called extracellular field potentials (EFPs). This noninvasive tool allows researchers to investigate key parameters such as EFP amplitude, duration, and arrhythmias. MEA is progressively considered the gold standard for high-throughput in vitro electrophysiological evaluation, particularly for cardiac disease modeling and cardiac toxicity assessment.

Heterologous Expression of an Unusual Ketosynthase, SxtA, Leads to Production of Saxitoxin Intermediates in Escherichia coli.

Paralytic shellfish toxins (PSTs) are neurotoxic alkaloids produced by freshwater cyanobacteria and marine dinoflagellates. Due to their antagonism of voltage-gated sodium channels in excitable cells, certain analogues are of significant pharmacological interest. The biosynthesis of the parent compound, saxitoxin, is initiated with the formation of 4-amino-3-oxo-guanidinoheptane (ethyl ketone) by an unusual polyketide synthase-like enzyme, SxtA. We have heterologously expressed SxtA from Raphidiopsis raciborskii T3 in Escherichia coli and analysed its activity in vivo. Ethyl ketone and a truncated analogue, methyl ketone, were detected by HPLC-ESI-HRMS analysis, thus suggesting that SxtA has relaxed substrate specificity in vivo. The chemical structures of these products were further verified by tandem mass spectrometry and labelled-precursor feeding with [guanidino-15 N2 ] arginine and [1,2-13 C2 ] acetate. These results indicate that the reactions catalysed by SxtA could give rise to multiple PST variants, including analogues of ecological and pharmacological significance.

Clinical Importance of the Human Umbilical Artery Potassium Channels.

Potassium (K+) channels are usually predominant in the membranes of vascular smooth muscle cells (SMCs). These channels play an important role in regulating the membrane potential and vessel contractility—a role that depends on the vascular bed. Thus, the activity of K+ channels represents one of the main mechanisms regulating the vascular tone in physiological and pathophysiological conditions. Briefly, the activation of K+ channels in SMC leads to hyperpolarization and vasorelaxation, while its inhibition induces depolarization and consequent vascular contraction. Currently, there are four different types of K+ channels described in SMCs: voltage-dependent K+ (KV) channels, calcium-activated K+ (KCa) channels, inward rectifier K+ (Kir) channels, and 2-pore domain K+ (K2P) channels. Due to the fundamental role of K+ channels in excitable cells, these channels are promising therapeutic targets in clinical practice. Therefore, this review discusses the basic properties of the various types of K+ channels, including structure, cellular mechanisms that regulate their activity, and new advances in the development of activators and blockers of these channels. The vascular functions of these channels will be discussed with a focus on vascular SMCs of the human umbilical artery. Then, the clinical importance of K+ channels in the treatment and prevention of cardiovascular diseases during pregnancy, such as gestational hypertension and preeclampsia, will be explored.

Isoform-specific regulation of HCN4 channels by a family of endoplasmic reticulum proteins

Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary proteins. Here, we report the discovery of a family of endoplasmic reticulum (ER) transmembrane proteins that associate with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, and Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. Despite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depolarizing shift in the voltage dependence of HCN4 in response to the binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage dependence in the absence of cAMP. The mechanisms of action of LRMP and IRAG are independent of trafficking and cAMP binding, and they are specific to the HCN4 isoform. We also found that IRAG is highly expressed in the mouse sinoatrial node where computer modeling predicts that its presence increases HCN4 current. Our results suggest important roles for LRMP and IRAG in the regulation of cellular excitability, as tools for advancing mechanistic understanding of HCN4 channel function, and as possible scaffolds for coordination of signaling pathways.

Paradoxical effects on voltage-gated Na+conductance in adrenal chromaffin cells by twin vs single high intensity nanosecond electric pulses.

We previously reported that a single 5 ns high intensity electric pulse (NEP) caused an E-field-dependent decrease in peak inward voltage-gated Na+ current (INa) in isolated bovine adrenal chromaffin cells. This study explored the effects of a pair of 5 ns pulses on INa recorded in the same cell type, and how varying the E-field amplitude and interval between the pulses altered its response. Regardless of the E-field strength (5 to 10 MV/m), twin NEPs having interpulse intervals ≥ than 5 s caused the inhibition of TTX-sensitive INa to approximately double relative to that produced by a single pulse. However, reducing the interval from 1 s to 10 ms between twin NEPs at E-fields of 5 and 8 MV/m but not 10 MV/m decreased the magnitude of the additive inhibitory effect by the second pulse in a pair on INa. The enhanced inhibitory effects of twin vs single NEPs on INa were not due to a shift in the voltage-dependence of steady-state activation and inactivation but were associated with a reduction in maximal Na+ conductance. Paradoxically, reducing the interval between twin NEPs at 5 or 8 MV/m but not 10 MV/m led to a progressive interval-dependent recovery of INa, which after 9 min exceeded the level of INa reached following the application of a single NEP. Disrupting lipid rafts by depleting membrane cholesterol with methyl-β-cyclodextrin enhanced the inhibitory effects of twin NEPs on INa and ablated the progressive recovery of this current at short twin pulse intervals, suggesting a complete dissociation of the inhibitory effects of twin NEPs on this current from their ability to stimulate its recovery. Our results suggest that in contrast to a single NEP, twin NEPs may influence membrane lipid rafts in a manner that enhances the trafficking of newly synthesized and/or recycling of endocytosed voltage-gated Na+ channels, thereby pointing to novel means to regulate ion channels in excitable cells.

Paralytic shellfish toxin biosynthesis in cyanobacteria and dinoflagellates: A molecular overview

Paralytic shellfish toxins (PSTs) are a group of water soluble neurotoxic alkaloids produced by two different kingdoms of life, prokaryotic cyanobacteria and eukaryotic dinoflagellates. Owing to the wide distribution of these organisms, these toxic secondary metabolites account for paralytic shellfish poisonings around the world. On the other hand, their specific binding to voltage-gated sodium channels makes these toxins potentially useful in pharmacological and toxicological applications. Much effort has been devoted to the biosynthetic mechanism of PSTs, and gene clusters encoding 26 proteins involved in PST biosynthesis have been unveiled in several cyanobacterial species. Functional analysis of toxin genes indicates that PST biosynthesis in cyanobacteria is a complex process including biosynthesis, regulation, modification and export. However, less is known about the toxin biosynthesis in dinoflagellates owing to our poor understanding of the massive genome and unique chromosomal characteristics [1]. So far, few genes involved in PST biosynthesis have been identified from dinoflagellates. Moreover, the proteins involved in PST production are far from being totally explored. Thus, the origin and evolution of PST biosynthesis in these two kingdoms are still controversial. In this review, we summarize the recent progress on the characterization of genes and proteins involved in PST biosynthesis in cyanobacteria and dinoflagellates, and discuss the standing evolutionary hypotheses concerning the origin of toxin biosynthesis as well as future perspectives in PST biosynthesis. Scientific questionParalytic shellfish toxins (PSTs) are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells and result in paralytic shellfish poisonings (PSPs) around the world. Two different kingdoms of life, cyanobacteria and dinoflagellates are able to produce PSTs. However, in contrast with cyanobacteria, our understanding of PST biosynthesis in dinoflagellates is extremely limited owing to their unique features. The origin and evolution of PST biosynthesis in these two kingdoms are still controversial. Technical significanceHigh-throughput omics technologies, such as genomics, transcriptomics and proteomics provide powerful tools for the study of PST biosynthesis in cyanobacteria and dinoflagellates, and have shown their powerful potential with regard to revealing genes and proteins involved in PST biosynthesis in two kingdoms. Scientific significanceThis review summarizes the recent progress in PST biosynthesis in cyanobacteria and dinoflagellates with focusing on the novel insights from omics technologies, and discusses the evolutionary relationship of toxin biosynthesis genes between these two kingdoms.

Novel perspectives in cancer therapy: Targeting ion channels.

By controlling ion fluxes at multiple time scales, ion channels shape rapid cell signals, such as action potential and synaptic transmission, as well as much slower processes, such as mitosis and cell migration. As is currently increasingly recognized, a variety of channel types are involved in cancer hallmarks, and regulate specific stages of neoplastic progression. Long-term in vitro work has established that inhibition of these ion channels impairs the growth of cancer cells. Recently, these studies have been followed up in vivo, hence revealing that ion channels constitute promising pharmacological targets in oncology. The channel proteins can be often accessed from the extracellular milieu, which allows use of lower drug doses and decrease untoward toxicity. However, because of the central physiological roles exerted by ion channels in excitable cells, other types of side effects may arise, the gravest of which is cardiac arrhythmia. A paradigmatic case is offered by Kv11.1 (hERG1) channels. HERG1 blockers attenuate the progression of both hematologic malignancies and solid tumors, but may also lead to the lengthening of the electrocardiographic QT interval, thus predisposing the patient to ventricular arrhythmias. These side effects can be avoided by specifically inhibiting the channel isoforms which are highly expressed in certain tumors, such as Kv11.1B and the neonatal forms of voltage-gated Na(+) channels. Preclinical studies are also being explored in breast and prostate cancer (targeting voltage-gated Na(+) channels), and gliomas (targeting CLC-3). Overall, the possible approaches to improve the efficacy and safety of ion channel targeting in oncology include: (1) the development of specific inhibitors for the channel subtypes expressed in specific tumors; (2) drug delivery into the tumor by using antibodies or nanotechnology-based approaches; (3) combination regimen therapy and (4) blocking specific conformational states of the ion channel. We believe that expanding this relatively neglected field of oncology research might lead to unforeseen therapeutic benefits for cancer patients.

An Analytical Method for the Quantification of hERG1 Channel Gene Expression in Human Colorectal Cancer

Cancer molecular investigation revealed a huge molecular heterogeneity between different types of cancers as well as among cancer patients affected by the same cancer type. This implies the necessity of a personalized approach for cancer diagnosis and therapy, on the basis of the development of standardized protocols to facilitate the application of molecular techniques in the clinical decision-making process. Ion channels encoding genes are acquiring increasing relevance in oncological translational studies, representing new candidates for molecular diagnostic and therapeutic purposes. Hence, the development of molecular protocols for the quantification of ion channels encoding genes in tumor specimens may have relevance for diagnostic and prognostic investigation. Two main hindrances must be overcome for these purposes: the use of formalin-fixed and paraffin-embedded samples for gene expression analysis and the physiological expression of ion channels in excitable cells, potentially present in the tumor sample. We here propose a method for hERG1 gene quantification in colorectal cancer samples in both cryopreserved and formalin-fixed and paraffin-embedded samples. An analytical method was developed to estimate hERG1 gene expression exclusively in epithelial cancer cells. Indeed, we found that the hERG1 gene was expressed at significant levels by myofibroblasts present in the tumor stroma. This method was based on the normalization on a smooth muscle-myofibroblast-specific gene, MYH11, with no need of microdissection. By applying this method, hERG1 expression turned out to correlate with VEGF-A expression, confirming previous immunohistochemical data.

Tuning voltage-gated channel activity and cellular excitability with a sphingomyelinase

Voltage-gated ion channels generate action potentials in excitable cells and help set the resting membrane potential in nonexcitable cells like lymphocytes. It has been difficult to investigate what kinds of phospholipids interact with these membrane proteins in their native environments and what functional impacts such interactions create. This problem might be circumvented if we could modify specific lipid types in situ. Using certain voltage-gated K+ (KV) channels heterologously expressed in Xenopus laevis oocytes as a model, our group has shown previously that sphingomyelinase (SMase) D may serve this purpose. SMase D is known to remove the choline group from sphingomyelin, a phospholipid primarily present in the outer leaflet of plasma membranes. This SMase D action lowers the energy required for voltage sensors of a KV channel to enter the activated state, causing a hyperpolarizing shift of the Q-V and G-V curves and thus activating them at more hyperpolarized potentials. Here, we find that this SMase D effect vanishes after removing most of the voltage-sensor paddle sequence, a finding supporting the notion that SMase D modification of sphingomyelin molecules alters these lipids’ interactions with voltage sensors. Then, using SMase D to probe lipid–channel interactions, we find that SMase D not only similarly stimulates voltage-gated Na+ (NaV) and Ca2+ channels but also markedly slows NaV channel inactivation. However, the latter effect is not observed in tested mammalian cells, an observation highlighting the profound impact of the membrane environment on channel function. Finally, we directly demonstrate that SMase D stimulates both native KV1.3 in nonexcitable human T lymphocytes at their typical resting membrane potential and native NaV channels in excitable cells, such that it shifts the action potential threshold in the hyperpolarized direction. These proof-of-concept studies illustrate that the voltage-gated channel activity in both excitable and nonexcitable cells can be tuned by enzymatically modifying lipid head groups.

The Electric Fingerprint of Membrane Voltage Sensor Domains

Voltage-sensor domains (VSDs) are electrically-charged constructs controlling the voltage-dependent activity of ion channels in excitable cells. Four packed transmembrane (TM) helices, S1 through S4, form the domain in which S4 contains 4 to 7 positively charged basic amino acids, mostly arginines. VSDs operate essentially by transferring the S4 charges across the transmembrane electric field (E), giving rise to the observable Q, the so-called “gating charge”. Mostly supported by structure-function studies on voltage-gated potassium (Kv) channels, a focused E has been identified as one key electric property of the VSD machinery. The recent increasing availability of other VSD-containing ion channel structures, including the x-ray structures for the NavAb and NavRh voltage-gated Na+ channels, provides us with the opportunity to extend the structure-based investigation of the domain electrostatic properties over a larger set of distinct conformations and isoforms. Using all-atom MD simulations in combination with electrostatic calculations, founded on an energetic formalism, we show that, over the entire set of available VSD structures, a specific hydration of the voltage sensor focuses E over a narrow TM region across the domain, at the vicinity of the so-called catalytic center. Furthermore, its focalization and shape is largely preserved over distinct conformations of the construct. Our results support that a focused and conformation-independent TM field is a robust electric feature of the VSD machinery, despite sequence variations or local structural modifications of the domain. This electric fingerprint seems to favor a highly conserved sensing mechanism for VSDs over the large family of voltage-gated cation channels.

Conus Venom Peptide Pharmacology

Conopeptides are a diverse group of recently evolved venom peptides used for prey capture and/or defense. Each species of cone snails produces in excess of 1000 conopeptides, with those pharmacologically characterized (≈ 0.1%) targeting a diverse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage- or ligand-gated ion channels, providing valuable research tools for the dissection of the role played by specific ion channels in excitable cells. It is noteworthy that many of these targets are found to be expressed in pain pathways, with several conopeptides having entered the clinic as potential treatments for pain [e.g., pyroglutamate1-MrIA (Xen2174)] and one now marketed for intrathecal treatment of severe pain [ziconotide (Prialt)]. This review discusses the diversity, pharmacology, structure-activity relationships, and therapeutic potential of cone snail venom peptide families acting at voltage-gated ion channels (ω-, μ-, μO-, δ-, ι-, and κ-conotoxins), ligand-gated ion channels (α-conotoxins, σ-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (ρ-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (χ-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and α-conotoxins. Expanding the discovery of new bioactives using proteomic/transcriptomic approaches combined with high-throughput platforms and better defining conopeptide structure-activity relationships using relevant membrane protein crystal structures are expected to grow the already significant impact conopeptides have had as both research probes and leads to new therapies.

Perturbation of Rat Heart Plasma Membrane Fluidity Due to Metabolites of Permethrin Insecticide

Due to increased global use, acute and chronic exposures to pyrethroid insecticides in humans are of clinical concern. Pyrethroids have a primary mode of action that involves interference with the sodium and calcium channels in excitable cells, which may include cardiac myocytes. Here, we investigated the possible cardiac toxicity of permethrin metabolites (METP), 3-phenoxy-benzyl alcohol (3PBA), 3-phenoxy-benzaldehyde (3PBALD), and 3-phenoxybenzoic acid (3PBACID). Plasma membrane fluidity, polarity, lipid, and protein oxidation were studied in isolated rat heart cells. Laurdan was chosen as probe to detect the lateral mobility and polarity of its environment and thus water penetration into the hydrophobic part of the bilayer, while 1,6-diphenyl-1,3,5-hexatriene permits to measure changes in fluidity in the inner part of the bilayer. Results show that METP can change membrane fluidity at different depths of the bilayer according to their partition coefficient. Consequently, 3PBA, at all concentration used, decreases membrane fluidity and polarity in the hydrophilic-hydrophobic region of the bilayer, and similar effect was observed with 20μM 3PBALD or 10 or 20μM 3 PBACID. Membrane dynamics in the hydrophobic core resulted decreased by 3PBALD, while it was increased by 20μM 3PBACID. All METP increase protein and lipid oxidation, and the peroxidative lipid damage decreases with the type of METP produced during the transformation pathway from parent compound to 3PBACID. Consequently, 3PBA induced the highest lipid peroxidation, while 3PBACID was the stronger inducer of protein damage.

Persistent membranous cross correlations due to the multiplicity of gates in ion channels

Ion channels in excitable cells reveal spontaneous intermittent opening and closing. As the membrane area reduces, this stochasticity enables spontaneous firing and elevates the cell's ability to fire at weaker stimuli. A multiple number of gates are accommodated in each individual ion channel. Here we investigate the possible impact of that gate multiplicity on the cell's function specifically when the membrane area is of limited size. It is shown that a non-trivially persistent correlation then takes place between the transmembrane voltage fluctuations (also between the fluctuations in the gating variables) and the component of open channel fluctuations attributed to the above gate multiplicity. This cross correlation persistency is found to be playing a major augmentative role in the elevation of the cell's excitability and spontaneous firing; without the persistency, the cell would be much less excitable. The cross correlation persistency is also found to enhance spike coherence. The stochastic Hodgkin-Huxley equations, put forward by Fox and Lu, are addressed in the context of their recognized failure to produce accurate enough statistics of spike generation. Our results indicate that the major source of that inaccuracy is the incapability of the stochastic Hodgkin-Huxley description to reflect the above cross correlation persistency.

Errors in the measurement of voltage-activated ion channels in cell-attached patch-clamp recordings

Patch-clamp recording techniques have revolutionized understanding of the function and sub-cellular location of ion channels in excitable cells. The cell-attached patch-clamp configuration represents the method of choice to describe the endogenous properties of voltage-activated ion channels in the axonal, somatic and dendritic membrane of neurons, without disturbance of the intracellular milieu. Here, we directly examine the errors associated with cell-attached patch-clamp measurement of ensemble ion channel activity. We find for a number of classes of voltage-activated channels, recorded from the soma and dendrites of neurons in acute brain-slices and isolated cells, that the amplitude and kinetics of ensemble ion channel activity recorded in cell-attached patches is significantly distorted by transmembrane voltage changes generated by the flow of current through the activated ion channels. We outline simple error–correction procedures that allow a more accurate description of the density and properties of voltage-activated channels to be incorporated into computational models of neurons.

Activity of BKCa Channel Is Modulated by Membrane Cholesterol Content and Association with Na+/K+-ATPase in Human Melanoma IGR39 Cells

Interaction of large conductance Ca(2+)- and voltage-activated K(+) (BK(Ca)) channels with Na(+)/K(+)-ATPase, caveolin-1, and cholesterol was studied in human melanoma IGR39 cells. Functional BK(Ca) channels were enriched in caveolin-rich and detergent-resistant membranes, i.e. rafts, and blocking of the channels by a specific BK(Ca) blocker paxilline reduced proliferation of the cells. Disruption of rafts by selective depletion of cholesterol released BK(Ca) channels from these domains with a consequent increase in their activity. Consistently, cholesterol enrichment of the cells increased the proportion of BK(Ca) channels in rafts and decreased their activity. Immunocytochemical analysis showed that BK(Ca) channels co-localize with Na(+)/K(+)-ATPase in a cholesterol-dependent manner, thus suggesting their co-presence in rafts. Supporting this, ouabain, a specific blocker of Na(+)/K(+)-ATPase, inhibited BK(Ca) whole-cell current markedly in control cells but not in cholesterol-depleted ones. This inhibition required the presence of external Na(+). Collectively, these data indicate that the presence of Na(+)/K(+)-ATPase in rafts is essential for efficient functioning of BK(Ca) channels, presumably because the pump maintains a low intracellular Na(+) proximal to the BK(Ca) channel. In conclusion, cholesterol could play an important role in cellular ion homeostasis and thus modulate many cellular functions and cell proliferation.

The role of TRPM4 in ventricular cardiomyocytes

TRPM4 is a Ca2+ activated non‐selective cation channel. Together with TRPM5 it's the only molecular candidate for this class of ion channels to date. Both genes are members of the Transient Receptor Potential (TRP) family of ion channels. We have shown previously, using Trpm4−/− mice, that this channel plays a critical role in setting the membrane potential, and consequently the driving force for Ca2+ entry in mast cells, which determines ultimately the strength of an allergic reaction. The role of CAN channels in excitable cells however is unclear. In this study we tested whether TRPM4 plays a role in cardiac muscle. We used the analysis of pressure‐volume loops to determine contractility properties in vivo. These results were confirmed in isolated papillary muscle. To determine the role of TRPM4 in cardiomyocytes, we used sharp intracellular micro‐electrodes to measure membrane potential in paced LV papillary muscle. We are currently analyzing the consequences of TRPM4 ablation for cellular signaling in isolated cardiomyocytes using the patch clamp technique.

Cellular prostheses: functional abiotic nanosystems to probe, manipulate, and endow function in live cells

A class of nanoscale (∼1–10 nm) structures designed to probe, manipulate, or endow function by direct interfacing with live cells is considered. Such a concept of cellular-level prostheses is illustrated via the example of light-activated nanoscale photodiodes capable of creating local electric fields that modulate existing voltage-gated ion channels in excitable cells. The dynamics of the membrane potential modulation by such photovoltaic functional abiotic nanosystems (PV-FANs) is modeled through an appropriate equivalent circuit. The feasibility of exceeding the typical ∼10 mV depolarization threshold for activating the action potentials is examined. In view of the continuing advances in the ability to design, synthesize, and characterize abiotic nanoscale systems that can provide desired function, several approaches to the implementation of PV-FANs are discussed. The FANs as “cellular prostheses” can provide a variety of functions in response to different stimuli and represent a paradigm-changing opportunity at the frontiers of nanomedicine. From the Clinical Editor A class of nanoscale (~1-10nm) structures designed to probe, manipulate, or endow live cell functions is demonstrated in this work. More specifically, light-activated nanoscale photodiodes were found capable of creating local electric fields that modulate existing voltage gated ion channels in excitable cells, thus allowing the generation of action potentials in excitable cells via external light stimulus in a controlled fashion.