Identical Biophysical Properties Research Articles

Mechanosensor transient receptor potential vanilloid member 4 (TRPV4) regulates mouse cholangiocyte secretion and bile formation.

Mechanosensitive signaling has emerged as a mechanism for the regulation of cholangiocyte transport and bile formation. The mechanical effect of fluid-flow, or shear, at the apical membrane of cholangiocytes regulates secretion through a process involving increases in [Ca2+]i and activation of Ca2+-activated Cl- channels. However, the initiating steps translating shear force to increases in intracellular calcium concentration ([Ca2+]i) are unknown. Transient receptor potential vanilloid member 4 (TRPV4), a nonselective cation channel present in the apical membrane of cholangiocytes, has been proposed as a potential mechanosensor. The aim of the present studies was to determine the potential role of TRPV4 in initiating mechanosensitive signaling in response to fluid-flow in cholangiocytes. TRPV4 expression was confirmed in both small and large mouse cholangiocytes. Exposure of cells to either fluid flow or specific TRPV4 pharmacological agonists rapidly increased both [Ca2+]i and membrane cation currents. Both flow- and agonist-stimulated currents displayed identical biophysical properties and were inhibited in the presence of TRPV4 antagonists or in cells after transfection with TRPV4 small interfering RNA. Transfection of mouse cholangiocytes with a TRPV4-enhanced green fluorescent protein construct increased the expression of TRPV4 and the magnitude of flow-stimulated currents. A specific TRPV4 agonist significantly increased the biliary concentration of ATP and bile flow in live mice when administered intravenously and increased ATP release from cholangiocyte monolayers when applied exogenously. The findings are consistent with a model in which activation of cholangiocyte TRPV4 translates shear force into an acute rise in membrane cation permeability, [Ca2+]i, ATP release, and bile flow. Understanding the role of mechanosensitive transport pathways may provide novel insights to modulate bile flow for the treatment of cholestatic liver disorders.NEW & NOTEWORTHY These studies functionally characterize TRPV4 as a mechanosensitive channel in mouse cholangiocytes. By mediating a rapid rise in intracellular Ca2+, necessary for Ca2+-dependent secretion, TRPV4 represents a mechanosensor responsible for translating fluid flow into intracellular signaling and biliary secretion. Furthermore, intravenous infusion of a specific TRPV4 agonist increases bile flow in live mice. Understanding the role of TRPV4 in mechanosensitive transport pathways may provide novel insights to modulate bile flow during cholestasis.

Regulation of the CaV3.2 calcium channels in health and disease Regulácia CaV3.2 vápnikových kanálov v zdraví a chorobe

Abstract Family of T-type or low-voltage activated calcium channels consists of three members: CaV3.1, CaV3.2, and CaV3.3. CaV3.2 channel has almost identical biophysical properties as the CaV3.1 channel, but is distinguished by a specific tissue expression profile and a prominent role in several pathologies, including neuropathic pain, epilepsy, and dysregulation of cardiac rhythm. Further, it may be involved in phenotype of autism spectrum disorders, and amyotrophic lateral sclerosis. It represents a promising target for future pharmacotherapies.

Network-level effects of optogenetic stimulation in a computer model of macaque primary motor cortex

Optogenetics is a potent tool for performing precise perturbations to ongoing cortical dynamics in behaving primates. However, only small numbers of neurons can be recorded simultaneously. In this work, we present a biomimetic spiking network model of macaque primary motor cortex (M1) in order to explore network-level effects of optogenetic stimulation – namely, how synaptic connections modulate the optogenetic stimulus response, and how optogenetic stimulation affects interlaminar information flow. Experimental data were recorded from M1 in a male macaque. Optogenetic stimulation targeted excitatory neurons, likely preferentially affecting deeper layers, via the excitatory opsin C1V1TT, with either continuous (200 ms duration) or periodic (20, 40, or 80 Hz) pulses of green light (561 nm) [1]. The network model consisted of 24,800 spiking Izhikevich neurons [2], consisting of regular-firing and bursting pyramidal neurons and fast-spiking and low-threshold-spiking interneurons, with connectivities and proportions of each cell type across each cortical layer drawn from empirical mammalian M1 literature. Opsin channel properties were based on empirical estimates. The network model was calibrated to reproduce experimentally observed firing rates and dynamics. Experimentally, optogenetic stimulation was found to increase firing rates by up to 300 spikes/sec, with higher firing rates observed close to the optrode; firing returned to baseline values between 4 and 5 mm laterally from the optrode. A similar response was observed in the model. Manipulating the strength of synaptic connectivity in the model elucidated several noteworthy aspects of the response. First, the decrease in peak firing rates very near the optrode appears to be due to increased recruitment of inhibitory neurons. Second, even though all excitatory neurons in the model had identical biophysical properties and received identical current input from the opsin, heterogeneities in connectivity were sufficient to account for the broad range of observed firing rates. Third, all of the high-firing excitatory cells (>100 spikes/sec) were opsin-expressing; no indirectly activated excitatory cells fired above 60 spikes/sec. Applying spectral Granger causality to the LFPs produced by the different cortical layers of the model showed that the strongest projection without stimulation was from layer 2/3 to layer 5A, consistent with the hypothesis that descending excitation is the primary driver of dynamics in the motor cortex. Strong Granger causality from layer 5A to layer 5B was also observed. Across all layer pairs, Granger causality showed a pronounced peak in the mu rhythm band (~9 Hz), with a small, broad bump in the gamma band (~40 Hz) observed in pathways from layer 2/3 to other layers. Optogenetic stimulation in the model increased Granger causality from layer 5 to other layers in a narrow band near the stimulation frequency. It also increased the amplitude of Granger causality from layer 2/3 to other layers in the mu rhythm band, while decreasing it in the gamma band. In summary, this work demonstrates that (1) synaptic connections play an important role in determining the network-level response to optogenetic stimulation, and (2) optogenetic stimulation may be used to enhance and suppress information flow in particular frequency bands and between particular cortical layers.

Impact of neuronal heterogeneity on correlated colored noise-induced synchronization

Synchronization plays an important role in neural signal processing and transmission. Many hypotheses have been proposed to explain the origin of neural synchronization. In recent years, correlated noise-induced synchronization has received support from many theoretical and experimental studies. However, many of these prior studies have assumed that neurons have identical biophysical properties and that their inputs are well modeled by white noise. In this context, we use colored noise to induce synchronization between oscillators with heterogeneity in both phase-response curves and frequencies. In the low noise limit, we derive novel analytical theory showing that the time constant of colored noise influences correlated noise-induced synchronization and that oscillator heterogeneity can limit synchronization. Surprisingly, however, heterogeneous oscillators may synchronize better than homogeneous oscillators given low input correlations. We also find resonance of oscillator synchronization to colored noise inputs when firing frequencies diverge. Collectively, these results prove robust for both relatively high noise regimes and when applied to biophysically realistic spiking neuron models, and further match experimental recordings from acute brain slices.

Alternative Splicing Regulates Kv3.1 Polarized Targeting to Adjust Maximal Spiking Frequency

Synaptic inputs received at dendrites are converted into digital outputs encoded by action potentials generated at the axon initial segment in most neurons. Here, we report that alternative splicing regulates polarized targeting of Kv3.1 voltage-gated potassium (Kv) channels to adjust the input-output relationship. The spiking frequency of cultured hippocampal neurons correlated with the level of endogenous Kv3 channels. Expression of axonal Kv3.1b, the longer form of Kv3.1 splice variants, effectively converted slow-spiking young neurons to fast-spiking ones; this was not the case for Kv1.2 or Kv4.2 channel constructs. Despite having identical biophysical properties as Kv3.1b, dendritic Kv3.1a was significantly less effective at increasing the maximal firing frequency. This suggests a possible role of channel targeting in regulating spiking frequency. Mutagenesis studies suggest the electrostatic repulsion between the Kv3.1b N/C termini, created by its C-terminal splice domain, unmasks the Kv3.1b axonal targeting motif. Kv3.1b axonal targeting increased the maximal spiking frequency in response to prolonged depolarization. This finding was further supported by the results of local application of channel blockers and computer simulations. Taken together, our studies have demonstrated that alternative splicing controls neuronal firing rates by regulating the polarized targeting of Kv3.1 channels.

Regulation of TMEM16A by a novel mechanism involving PKC in biliary epithelium

TMEM16A is a Ca2+‐activated Cl− channel in the apical membrane of biliary epithelial cells (BECs), and contributes to biliary secretion and bile formation [Dutta et al. JBC, 2010]. Extracellular nucleotides activate TMEM16A via binding P2 receptors and increasing [Ca2+]i. While PKC contributes to ATP‐mediated secretion in BECs, the role in TMEM16A regulation is unknown.AimTo determine if PKC contributes to Cl− transport in BECs through regulation of TMEM16A.MethodsStudies were performed in human biliary Mz‐cha‐1 cells. Cl− currents were measured by whole‐cell patch clamp.ResultsTMEM16A Cl− currents were activated by exposure to extracellular ATP or increasing Ca2+ in the patch pipette. Currents had a reversal potential of 0 mV, time‐dependent activation and outward rectification. Currents were significantly inhibited by anti‐TMEM16A siRNA. In the presence of the PKC inhibitors Chelerithrine or Gö6976 Cl− currents were significantly inhibited. Conversely, activation by PMA resulted in large Cl− currents. Intracellular dialysis with recombinant PKCα activated Cl− currents with identical biophysical properties to TMEM16A.ConclusionPKCα couples ATP‐mediated signaling to TMEM16A activation in BECs. Modulation of kinase signaling pathways may represent a target for increasing biliary secretion and bile formation.[Studies are supported by AASLD/ALF Liver Scholar Award 2010, Children's Medical Center Foundation grant (Dutta), and NIH NIDDK DK078587]

Highly Ca2+-selective TRPM Channels Regulate IP3-dependent Oscillatory Ca2+ Signaling in the C. elegans Intestine

Posterior body wall muscle contraction (pBoc) in the nematode Caenorhabditis elegans occurs rhythmically every 45–50 s and mediates defecation. pBoc is controlled by inositol-1,4,5-trisphosphate (IP3)–dependent Ca2+ oscillations in the intestine. The intestinal epithelium can be studied by patch clamp electrophysiology, Ca2+ imaging, genome-wide reverse genetic analysis, forward genetics, and molecular biology and thus provides a powerful model to develop an integrated systems level understanding of a nonexcitable cell oscillatory Ca2+ signaling pathway. Intestinal cells express an outwardly rectifying Ca2+ (ORCa) current with biophysical properties resembling those of TRPM channels. Two TRPM homologues, GON-2 and GTL-1, are expressed in the intestine. Using deletion and severe loss-of-function alleles of the gtl-1 and gon-2 genes, we demonstrate here that GON-2 and GTL-1 are both required for maintaining rhythmic pBoc and intestinal Ca2+ oscillations. Loss of GTL-l and GON-2 function inhibits IORCa ∼70% and ∼90%, respectively. IORCa is undetectable in gon-2;gtl-1 double mutant cells. These results demonstrate that (a) both gon-2 and gtl-1 are required for ORCa channel function, and (b) GON-2 and GTL-1 can function independently as ion channels, but that their functions in mediating IORCa are interdependent. IORCa, IGON-2, and IGTL-1 have nearly identical biophysical properties. Importantly, all three channels are at least 60-fold more permeable to Ca2+ than Na+. Epistasis analysis suggests that GON-2 and GTL-1 function in the IP3 signaling pathway to regulate intestinal Ca2+ oscillations. We postulate that GON-2 and GTL-1 form heteromeric ORCa channels that mediate selective Ca2+ influx and function to regulate IP3 receptor activity and possibly to refill ER Ca2+ stores.

Biophysical characterization of S100A8 and S100A9 in the absence and presence of bivalent cations

S100A8 and S100A9 are two proinflammatory molecules belonging to the S100 family of calcium-binding proteins. Common to all S100 proteins S100A8 and S100A9 form non-covalently associated complexes which have been shown to exhibit different functional properties. Besides dimerization, recent research is focused on the importance of higher oligomeric structures of S100 proteins induced by bivalent cations. While S100A8/S100A9-heterodimers are formed in the absence of calcium, tetramerization is strictly calcium-dependent. Heterodimer formation is not a simple process and our biophysical analyses (FRET, ESI-MS) demonstrate that simply mixing both subunits is not sufficient to induce complex formation. Steps of denaturation/renaturation are necessary for the recombinant complex to show identical biophysical properties as S100A8/S100A9 obtained from granulocytes. In addition to calcium both proteins are able to bind zinc with high affinity. Here we demonstrate for the first time by different biophysical methods (MALDI-MS, ESI-MS, fluorescence spectroscopy) that zinc-binding, like calcium, induces (S100A8/S100A9) 2-tetramers. Using mass spectrometric investigations we demonstrate that zinc triggers the formation of (S100A8/S100A9) 2-tetramers by zinc-specific binding sites rather than by interactions with calcium-specific EF-hands. The zinc-induced tetramer is structurally very similar to the calcium-induced tetramer. Thus, like calcium, zinc acts as a regulatory factor in S100A8/S100A9-dependent signaling pathways.

Regulation of an Outwardly Rectifying Chloride Conductance in Renal Epithelial Cells by External and Internal Calcium

We have used perforated patch clamp and Fura-2 microfluorescence measurements to study Ca(2+)-activated Cl- currents in cultured mouse renal inner medullary collecting duct cells (mIMCD-3). The conductance was spontaneously active under resting conditions and whole cell currents were time and voltage-independent with an outwardly rectifying current-voltage relationship. The channel blockers DIDS, niflumic acid, glybenclamide and NPPB reversibly decreased the basal currents, whereas the sulfhydryl agent, DTT produced an irreversible inhibition. Increasing or decreasing extracellular calcium produced parallel changes in the size of the basal currents. Variations in external Ca2+ were associated with corresponding changes in free cytosolic Ca2+ concentration. Increasing cytosolic Ca2+ by extracellular ATP or ionomycin, further enhanced Cl- conductance, with whole cell currents displaying identical biophysical properties to the basal currents. However, the agonist-stimulated currents were now increased by DTT exposure, but still inhibited by the other channel blockers. Using RT-PCR, three distinct mRNA transcripts belonging to the CLCA family of Ca(2+)-activated Cl- channel proteins were identified, two of which represent novel sequences. Whether different channels underlie the basal and agonist-stimulated currents in mIMCD-3 cells is unclear. Our findings establish a novel link between alterations in external and internal Ca2+ and the activity of Ca(2+)-activated Cl- transport in these cells.

Blocking adenylyl cyclase inhibits olfactory generator currents induced by "IP(3)-odors".

Vertebrate olfactory receptor neurons (ORNs) transduce odor stimuli into electrical signals by means of an adenylyl cyclase/cAMP second messenger cascade, but it remains widely debated whether this cAMP cascade mediates transduction for all odorants or only certain odor classes. To address this problem, we have analyzed the generator currents induced by odors that failed to produce cAMP in previous biochemical assays but instead produced IP(3) ("IP(3)-odors"). We show that in single salamander ORNs, sensory responses to "cAMP-odors" and IP(3)-odors are not mutually exclusive but coexist in the same cells. The currents induced by IP(3)-odors exhibit identical biophysical properties as those induced by cAMP odors or direct activation of the cAMP cascade. By disrupting adenylyl cyclase to block cAMP formation using two potent antagonists of adenylyl cyclase, SQ22536 and MDL12330A, we show that this molecular step is necessary for the transduction of both odor classes. To assess whether these results are also applicable to mammals, we examine the electrophysiological responses to IP(3)-odors in intact mouse main olfactory epithelium (MOE) by recording field potentials. The results show that inhibition of adenylyl cyclase prevents EOG responses to both odor classes in mouse MOE, even when "hot spots" with heightened sensitivity to IP(3)-odors are examined.

Spontaneously oscillating K+ channel activity in transformed Madin-Darby canine kidney cells.

Intracellular alkalinization is known to be associated with tumorigenic transformation. Besides phenotypical alterations alkali-transformed Madin-Darby canine kidney (MDCK) cells exhibit a spontaneously oscillating cell membrane potential (PD). Using single-channel patch clamp techniques, it was the aim of this study to identify the ion channel underlying the rhythmic hyperpolarizations of the PD. In the cell-attached patch configuration, we found that channel activity was oscillating. The frequency of channel oscillations is 1.1 +/- 0.1 min-1. At the peak of oscillatory channel activity, single-channel current was -2.7 +/- 0.05 pA, and in the resting state it was -1.95 +/- 0.05 pA. Given the single-channel conductance of 53 +/- 3 pS for inward (and of 27 +/- 5 pS for outward) current the difference of single-channel current amplitude corresponded to a hyperpolarization of approximately 14 mV. The channel is selective for K+ over Na+. Channel kinetics are characterized by one open and by three closed time constants. The channel is Ca2+ sensitive. Half maximal activation in the inside-out patch mode is achieved at a Ca2+ concentration of 10 mumol/liter. In addition, we also found a 13-pS K+ channel that shows no oscillatory activity in the cell-attached patch configuration and that was not Ca2+ sensitive. We conclude that the Ca(2+)-sensitive 53-pS K+ channel is underlying spontaneous oscillations of the PD. It has virtually identical biophysical properties as a Ca(2+)-sensitive K+ channel in nontransformed parent MDCK cells. Hence, alkali-induced transformation of MDCK cells did not affect the channel protein itself but its regulators thereby causing spontaneous fluctuations of the PD.

Human placental 3β-hydroxy-5-ene-steroid dehydrogenase and steroid 5→ 4-ene-isomerase: Purification from mitochondria and kinetic profiles, biophysical characterization of the purified mitochondrial and microsomal enzymes

In human placenta, 3β-hydroxy-5-ene-steroid dehydrogenase and steroid 5→ 4-ene-isomerase, an enzyme complex found in microsomes and mitochondria, synthesizes progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate. The dehydrogenase and isomerase activities of the mitochondrial enzyme were copurified (733-fold) using sequential cholate solubilization, ion exchange chromatography (DEAE-Toyopearl 650S), and hydroxylapatite chromatography (Bio-Gel HT). Enzyme homogeneity was demonstrated by a single protein band in SDS-polyacrylamide gel electrophoresis (monomeric M r = 41,000), gel filtration at constant specific enzyme activity (M r = 77,000), and a single NH 2-terminal sequence. Kinetic constants were determined for the oxidation of pregnenolone ( K m = 1.6 μM, V max = 48.6 nmol/min/mg) and dehydroepiandrosterone ( K m = 2.4 μM, V max = 48.5 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione ( K m = 9.3 μM, V max = 914.2 nmol/min/mg) and 5-androstene-3,17-dione ( K m = 27.6 μM, 0 V max = 888.4 nmol/min/mg. Mixed substrate studies showed that the dehydrogenase and isomerase activities utilize their respective pregnene and androstene substrates competitively. Dixon analysis demonstrated that the product steroids, progesterone and androstenedione, are competitive inhibitors of the C-21 and C-19 dehydrogenase activities. Enzyme purified from mitochondria and microsomes had similar kinetic profiles with respect to substrate utilization, product inhibition, and cofactor (NAD +) reduction (mean K m ± SD using C-19 and C-21 dehydrogenase substrates = 26.4 ± 0.8 μM, mean V max = 73.2 ± 1.3 nmol/min/mg). Pure enzyme from both organelles exhibited identical biophysical properties in terms of molecular weight and subunit composition, pH optima (pH 9.8, dehydrogenase; pH 7.5, isomerase), temperature optimum (37°C), stability in storage and solution, effects of divalent cations, and the single NH 2-terminal sequence of 27 amino acids. These results suggest that the mitochondrial and microsomal enzymes are the same protein localized in different organelles.