Unitary Conductance Research Articles

Differential regulation of Connexin50 and Connexin46 by PI3K signaling

Gap junction channels can modify their activity in response to cell signaling pathways. Here, we demonstrate that Connexin50 (Cx50) coupling, but not Connexin46 (Cx46), increased when co-expressed with a constitutively active p110α subunit of PI3K in Xenopus oocytes. In addition, inhibition of PI3K signaling by blocking p110α, or Akt, significantly decreased gap junctional conductance in Cx50 transfected HeLa cells, with no effect on Cx46. Alterations in coupling levels were not a result of reduced Cx50 unitary conductance, suggesting that changes in the number of active channels were responsible. These data indicate that Cx50 is specifically regulated by the PI3K signaling pathway.

Amiloride-insensitive sodium channels are directly regulated by actin cytoskeleton dynamics in human lymphoma cells

Sodium influx mediated by ion channels of plasma membrane underlies fundamental physiological processes in cells of blood origin. However, little is known about the single channel activity and regulatory mechanisms of sodium-specific channels in native cells. In the present work, we used different modes of patch clamp technique to examine ion channels involved in Na-transporting pathway in U937 human lymphoma cells. The activity of native non-voltage-gated sodium (NVGS) channels with unitary conductance of 10 pS was revealed in cell-attached, inside-out and whole-cell configurations. NVGS channel activity is directly controlled by submembranous actin cytoskeleton. Specifically, an activation of sodium channels in U937 cells in response to microfilament disassembly was demonstrated on single-channel and integral current level. Inside-out experiments showed that filament assembly on cytoplasmic membrane surface caused fast inactivation of the channels. Biophysical characteristics of NVGS channels in U937 cells were similar to that of epithelial sodium channels (ENaCs). However, we found that amiloride, a known inhibitor of DEG/ENaC, did not block NVGS channels in U937 cells. Whole-cell current measurements revealed no amiloride-sensitive component of membrane current. Our data show that cortical actin structures represent the main factor that controls the activity of amiloride-insensitive ENaC-like channels in human lymphoma cells.

O2 Dependent Contributions of Prostaglandins (PGI2 PGE2) to Exercise Hyperaemia in Young (Y) and Older (O) Men

We have shown that breathing 40% O2 and cyclooxygenase (COX) inhibition attenuate exercise hyperaemia to similar extents in Y and O men. PGI2 synthesis is generally attributed to endothelium and PGE2, to skeletal muscle fibres. Thus, in recreationally active Y (18‐25 yrs; n=12) and O (60‐76 yrs; n=12) males, we assayed venous efflux of PGs by ELISA following rhythmic and static handgrip contractions (60% MVC for 3 min, or exhaustion), during air breathing, 40%O2, and after COX blockade with aspirin (A) in a cross over design. In Y males, forearm vascular conductance (FVC) increased from 0.06±0.01 to 0.56±0.06 conductance units (CU) after rhythmic contractions in P/air, but to only 0.44±0.04*, 0.44±0.04* and 0.45±0.03* CU in P/O2, A/air and A/O2 (*P < 0.05 vs P/air). Meanwhile, PGI2 increased from 18.0±6.1 to 349.8±61.7 pg/dl/min in P/air, but to 94.3±26.5*, 44.7±18.4* and 50.8±11.2* pg/dl/min in P/O2, A/air and A/O2, while PGE increased from 20.5±6.6 to 559.5±133.4 pg/dl/min in P/air, but to 189.8±33.8*, 165.4±52.9* and 170.3±43.0* pg/dl/min in P/O2, A/air and A/O2. Similarly in O males, FVC increased from 0.08±0.02 to 0.64±0.06 CU in P/air after rhythmic contraction, but to 0.44±0.06*, 0.46±0.05* and 0.52±0.06* CU in P/O2, A/air and A/O2. Meanwhile, PGI2 increased from 14.4±6.8 to 284.4±43.4 in P/air but to 84.6±25.9*, 52.8±20.3* and 74.6±38.1* pg/dl/min in P/O2, A/air and A/O2, while PGE increased from 16.6±6.6 to 781.6±192.5 in P/air, but to 368.8±188.8*, 357.9±106.4* and 382.3±131.0* pg/dl/min in P/O2, A/air and A/O2. Static contractions yielded similar data. Thus, we propose that PGI2 and PGE2 are released in O2‐dependent ways from endothelium and muscle fibres respectively and contribute to exercise hyperaemia in both Y and O men.

High Activity of Large‐conductance Ca 2+ ‐activated K + channels Underlies Weak Myogenic Tone of Rat Mesenteric Arteries in Comparison of Cerebral Artery

Myogenic tone (MT) of resistance artery, vasoconstriction in response to increasing luminal pressure (Plum), stabilizes regional blood flow albeit the fluctuation of perfusion pressure. The levels of MT differ between vascular beds. Mechanosensitive depolarization of membrane potential and activation of voltage-gated Ca2+ channels (VOCCs) are cellular mechanisms for MT in smooth muscle cells (SMCs). Large-conductance Ca2+-activated K+ channel (BKCa) in SMCs may antagonize the contractile signals in MT. Using video-analysis of pressurized (60 mmHg) artery, we found that MT in mesenteric artery (MA) is significantly smaller than that of cerebral artery (CA). Iberiotoxin, a selective BKCa inhibitor, augmented the MT of MA by larger extent than CA. Patch clamp analysis showed that not only the BKCa current density but also unitary slope conductance of MASMC are higher than CASMCs. In addition, the frequency of spontaneous transient outward currents (STOCs) reflecting the BKCa activity in responses to Ca2+ sparks was higher in MASMCs. Voltage-gated K+ channel (Kv) activity was also higher in MASMCs. Although Ba2+-sensitive inwardly rectifying K+ current is larger in CASMCs, pretreatment with Ba2+did not induce significant augmentation of iberiotoxin sensitivity of MT in CA. Taken together, it is suggested that more frequent STOCs and larger average outward current via BKCa could explain the weak MT in MA.

TRPV3 Sparklets Mediate Endothelium‐Dependent Dilation of Cerebral Parenchymal Arterioles

Elementary Ca2+ influx events through single transient receptor potential vanilliod 4 (TRPV4) channels, called TRPV4 sparklets, mediate endothelium-dependent dilation of mesenteric arteries. TRPV3 channels are present in the cerebral artery endothelium and have a larger unitary conductance and Ca2+ permeability compared with TRPV4 channels. Therefore, we attempted to use total internal reflection fluorescent microscopy (TIRFM) to record TRPV3 sparklets from primary cerebral artery endothelial cells. Carvacrol, a monterpenoid compound derived from oregano, increased the frequency of TRPV3 sparklets in a concentration-dependent manner (EC50 = 8.5 µM). Carvacrol-induced TRPV3 sparklets were inhibited by the selective TRPV3 blocker isopentenyl pyrophosphate (IPP). TRPV3 sparklets have a greater unitary amplitude (ΔF/F0 = 0.20), spatial spread (0.74 µm2), and duration (70 ms) than TRPV4 sparklets, suggesting that TRPV3-mediated Ca2+ influx could have a robust influence on cerebrovascular tone. In pressure myograph experiments, carvacrol caused dilation of cerebral parenchymal arterioles that was blocked by IPP. Carvacrol-induced dilation was also inhibited by block of intermediate and small conductance Ca2+-activated K+ (IK and SK) channels. Together, these data suggest that TRPV3 sparklets cause dilation of cerebral parenchymal arterioles by activating SK and IK channels in the endothelium. Supported by HL091905.

The Residues in the First Extracellular Domain Play an Important Role in Transjunctional-Voltage Dependent Gating and Unitary Channel Conductance of Cx50 Gap Junction Channels

Gap junctions (GJ) are intercellular channels that connecting the cytoplasm of neighbor cells. GJ channels formed by Cx50 and Cx36 show drastic disparity in their unitary conductance (γj) and transjunctional voltage-dependent gating (Vj-gating), but the important underlying molecular domains/residues are not clear. Experimental evidence showed that residues in the first extracellular domain (E1) of Cx50 likely line the GJ channel pore and are important factors in determining γj and Vj-gating. We aligned the E1 sequence of Cx50 with that of Cx36 and found 10 different residues (4/10 residues involves a change in charge). We generated a chimera Cx50Cx36E1, in which the E1 of Cx50 was replaced by the E1 of Cx36, and 4 point mutations in E1 of Cx50 (where a charge change occurs, i.e. G46E, D51M, E62N and E68R). Dual patch clamp study on the homotypic GJ channels formed by the chimera or the point mutants in N2A cell pairs indicate that the Cx50Cx36E1 channel showed little change in the Vj-gating properties, but displayed a significantly reduced main single channel conductance (γj). Our studies on the point mutations of Cx50 showed that some of the mutants altered the Vj-gating properties and others changed the γj and/or the probabilities in different conducting states. Our study indicates that Cx50 E1 is an important domain in determining the Vj-gating properties and γj. Charge changes in different residues in the E1 between Cx50 and Cx36 showed different channel properties likely dictated by either their location in the pore structure or the nature of the mutation.

Effects of External and Internal Ca2+ on Unitary NMDA Receptor Properties

N-Methyl-D-aspartate (NMDA) receptors are glutamate- and glycine-gated channels widely expressed throughout the central nervous system. When active, they generate electric and biochemical signals by fluxing Na+ and Ca2+ into the post-synaptic cell. The NMDA receptor-mediated Ca2+ transient initiates internal cascades that result in synaptic plasticity and excitotoxicity. In turn, Ca2+ regulates channel activity directly and indirectly through allosteric and 2nd messenger mechanisms that are poorly understood. We investigated the effects of external and internal Ca2+ concentrations on recombinant GluN1/GluN2A (N1/2A) receptors using single-channel current recordings, statistical analysis, and kinetic modeling. Increasing concentrations of extracellular Ca2+ reduced the unitary channel conductance from 75.9±1.4 pS in 0 mM [Ca2+]e, to 55.2±1.6 pS in 1.8 mM [Ca2+]e, and to 11.8±0.7 pS in 75 mM [Ca2+]e. Importantly, receptors lacking the intracellular C-terminal domain (CTD) of the N1 subunit (N1Δ/2A) but not the 2A subunit (N1/2AΔ) exhibited higher unitary conductance in the absence of external Ca2+ (N1Δ/2A γ = 83.3±1.0 pS, N1/2AΔ γ = 79.2±1.4 pS). This is consistent with a role for the N1 CTD in setting channel conductance for Na+. However, in the presence of physiological external Ca2+ (1.8 mM), both exhibited N1Δ/2A (γ = 50.4±1.2 pS, p<0.05) and N1/2AΔ (γ = 60.8±2.0 pS, p<0.05) channels had a lower conductance relative to wild-type. Based on these novel results, we asked whether this Ca2+-dependent regulation of conductance depends on calmodulin binding to either the C0 or C1 cassettes of N1. Furthermore, we asked whether local Ca2+ influx through the NMDA receptor pore is sufficient for this regulation. Our results help to further unravel the Ca2+-dependent processes that control the properties of individual NMDA receptor channels.

The Role of the Ion Dehydration Process in Low and High Conductance K Channels

Potassium channels are known for exhibiting a wide conductance rate from 8.2 pS to 250 pS despite that selectivity filter is largely conserved. Some aspects about the relation between the charge distribution along to the pore and the conductance rate have been studied. In Shaker channel, a low conductance K-channel, one single point mutation of the P475D increases the 4-fold the maximal unitary conductance. At the same time, BK channels, a large conductance K-channel, has been demonstrated that a ring of negative charged residues in the intracellular entrance are key residues in the control of the maximal ion transport rate. Another main difference between low and high conductance K-channels is the size of the channel pore, BK has a much wider internal vestibule than Shaker. In order to understand at the molecular level the conduction process in both K+ channel classes, we performed molecular dynamics simulations using an applied external electric field to compare the structural properties of BK and Shaker channels (+300 mV, +600 mV and +800mV). Different ranges of currents were observed in Shaker and BK under the influence of the external electric field. At all the voltages tested, BK present the largest rate of ion translocation. Interestingly, K+ ions moving from the intracellular side up to the S4 site show a dehydration pattern, which is more efficient in BK channels. Other channel structural properties are discussed in the context of the dehydration process of the K ions in Shaker and BK.Acknowledgements: CINV is a Millennium Institute. R.S thanks CONICYT doctoral fellowship and project FONDECYT 1110430 (RL), 1131003 (FGN), 1120819 (DN) and proyecto Anillo ACT1107 (FGN)

Ethanol modulation of mammalian BK channels in excitable tissues: molecular targets and their possible contribution to alcohol-induced altered behavior.

In most tissues, the function of Ca2+- and voltage-gated K+ (BK) channels is modified in response to ethanol concentrations reached in human blood during alcohol intoxication. In general, modification of BK current from ethanol-naïve preparations in response to brief ethanol exposure results from changes in channel open probability without modification of unitary conductance or change in BK protein levels in the membrane. Protracted and/or repeated ethanol exposure, however, may evoke changes in BK expression. The final ethanol effect on BK open probability leading to either BK current potentiation or BK current reduction is determined by an orchestration of molecular factors, including levels of activating ligand (Ca2+i), BK subunit composition and post-translational modifications, and the channel's lipid microenvironment. These factors seem to allosterically regulate a direct interaction between ethanol and a recognition pocket of discrete dimensions recently mapped to the channel-forming (slo1) subunit. Type of ethanol exposure also plays a role in the final BK response to the drug: in several central nervous system regions (e.g., striatum, primary sensory neurons, and supraoptic nucleus), acute exposure to ethanol reduces neuronal excitability by enhancing BK activity. In contrast, protracted or repetitive ethanol administration may alter BK subunit composition and membrane expression, rendering the BK complex insensitive to further ethanol exposure. In neurohypophyseal axon terminals, ethanol potentiation of BK channel activity leads to a reduction in neuropeptide release. In vascular smooth muscle, however, ethanol inhibition of BK current leads to cell contraction and vascular constriction.

Human Slack Potassium Channel Mutations Increase Positive Cooperativity between Individual Channels

Disease-causing mutations in ion channels generally alter intrinsic gating properties such as activation, inactivation, and voltage dependence. We examined nine different mutations of the KCNT1 (Slack) Na(+)-activated K(+) channel that give rise to three distinct forms of epilepsy. All produced many-fold increases in current amplitude compared to the wild-type channel. This could not be accounted for by increases in the intrinsic open probability of individual channels. Rather, greatly increased opening was a consequence of cooperative interactions between multiple channels in a patch. The degree of cooperative gating was much greater for all of the mutant channels than for the wild-type channel, and could explain increases in current even in a mutant with reduced unitary conductance. We also found that the same mutation gave rise to different forms of epilepsy in different individuals. Our findings indicate that a major consequence of these mutations is to alter channel-channel interactions.

Super-resolution imaging reveals that loss of the C-terminus of connexin43 limits microtubule plus-end capture and NaV1.5 localization at the intercalated disc.

It is well known that connexin43 (Cx43) forms gap junctions. We recently showed that Cx43 is also part of a protein-interacting network that regulates excitability. Cardiac-specific truncation of Cx43 C-terminus (mutant 'Cx43D378stop') led to lethal arrhythmias. Cx43D378stop localized to the intercalated disc (ID); cell-cell coupling was normal, but there was significant sodium current (INa) loss. We proposed that the microtubule plus-end is at the crux of the Cx43-INa relation. Yet, specific localization of relevant molecular players was prevented due to the resolution limit of fluorescence microscopy. Here, we use nanoscale imaging to establish: (i) the morphology of clusters formed by the microtubule plus-end tracking protein 'end-binding 1' (EB1), (ii) their position, and that of sodium channel alpha-subunit NaV1.5, relative to N-cadherin-rich sites, and (iii) the role of Cx43 C-terminus on the above-mentioned parameters and on the location-specific function of INa. Super-resolution fluorescence localization microscopy in murine adult cardiomyocytes revealed EB1 and NaV1.5 as distinct clusters preferentially localized to N-cadherin-rich sites. Extent of co-localization decreased in Cx43D378stop cells. Macropatch and scanning patch clamp showed reduced INa exclusively at cell end, without changes in unitary conductance. Experiments in Cx43-modified HL1 cells confirmed the relation between Cx43, INa, and microtubules. NaV1.5 and EB1 localization at the cell end is Cx43-dependent. Cx43 is part of a molecular complex that determines capture of the microtubule plus-end at the ID, facilitating cargo delivery. These observations link excitability and electrical coupling through a common molecular mechanism.

Monovalent Cationic Channel Activity in the Inner Membrane of Nuclei from Skeletal Muscle Fibers

Nuclear ion channels remain among the least studied and biophysically characterized channels. Although considerable progress has been made in characterizing calcium release channels in the nuclear membrane, very little is known regarding the properties of nuclear monovalent cationic channels. Here, we describe a method to isolate nuclei from adult skeletal muscle fibers that are suitable for electrophysiological experiments. Using this approach, we show for the first time, to our knowledge, that a nuclear monovalent cationic channel (NMCC) is prominently expressed in the inner membrane of nuclei isolated from flexor digitorum brevis skeletal muscle fibers of adult mice. In isotonic 140 mM KCl, the skeletal muscle NMCC exhibits a unitary conductance of ∼160 pS and high, voltage-independent open probability. Based on single-channel reversal potential measurements, NMCCs are slightly more permeable to potassium ions over sodium (PK/PNa = 2.68 ± 0.21) and cesium (PK/PCs = 1.39 ± 0.03) ions. In addition, NMCCs do not permeate divalent cations, are inhibited by calcium ions, and demonstrate weak rectification in asymmetric Ca2+-containing solutions. Together, these studies characterize a voltage-independent NMCC in skeletal muscle, the properties of which are ideally suited to serve as a countercurrent mechanism during calcium release from the nuclear envelope.

Stochastic transport through carbon nanotubes in lipid bilayers and live cell membranes.

There is much interest in developing synthetic analogues of biological membrane channels with high efficiency and exquisite selectivity for transporting ions and molecules. Bottom-up and top-down methods can produce nanopores of a size comparable to that of endogenous protein channels, but replicating their affinity and transport properties remains challenging. In principle, carbon nanotubes (CNTs) should be an ideal membrane channel platform: they exhibit excellent transport properties and their narrow hydrophobic inner pores mimic structural motifs typical of biological channels. Moreover, simulations predict that CNTs with a length comparable to the thickness of a lipid bilayer membrane can self-insert into the membrane. Functionalized CNTs have indeed been found to penetrate lipid membranes and cell walls, and short tubes have been forced into membranes to create sensors, yet membrane transport applications of short CNTs remain underexplored. Here we show that short CNTs spontaneously insert into lipid bilayers and live cell membranes to form channels that exhibit a unitary conductance of 70-100picosiemens under physiological conditions. Despite their structural simplicity, these 'CNT porins' transport water, protons, small ions and DNA, stochastically switch between metastable conductance substates, and display characteristic macromolecule-induced ionic current blockades. We also show that local channel and membrane charges can control the conductance and ion selectivity of the CNT porins, thereby establishing these nanopores as a promising biomimetic platform for developing cell interfaces, studying transport in biological channels, and creating stochastic sensors.

Charge at the 46th residue of connexin 50 is crucial for the gap-junctional unitary conductance and transjunctional voltage-dependent gating.

Gap-junction (GJ) channels are twice the length of most membrane channels, yet they often have large unitary channel conductance (γj). What factors make this possibly the longest channel so efficient in passing ions are not fully clear. Here we studied the lens connexin (Cx) 50 GJs, which display one of the largest γj and the most sensitive transjunctional voltage-dependent gating (Vj gating) among all GJ channels. Introduction of charged residues into a putative pore-lining domain (the first transmembrane and the first extracellular loop border) drastically altered the apparent γj. Specifically, G46D and G46E increased the Cx50 γj from 201 to 256 and 293 pS, respectively and the G46K channel showed an apparent γj of only 20 pS. G46K also drastically altered Vj gating properties in homotypic G46K and heterotypic Cx50/G46K channels, causing an apparent loss of fast Vj-dependent gating transitions and leaving only loop gating transitions at the single channel current records. Both macroscopic and single channel currents of heterotypic Cx50/G46K channels showed a prominent rectification. Our homology structural models indicate that the pore surface electrostatic potentials are a dictating factor in determining the γj. Our data demonstrate, at the whole GJ channel level, a crucial role of the surface charge properties in the first transmembrane/first extracellular border domain in determining the efficiency of ion permeation and the Vj gating of Cx50 and possibly other GJ channels.

Extracellular Ca(2+) ions reduce NMDA receptor conductance and gating.

Brief intracellular Ca(2+) transients initiate signaling routines that direct cellular activities. Consequently, activation of Ca(2+)-permeable neurotransmitter-gated channels can both depolarize and initiate remodeling of the postsynaptic cell. In particular, the Ca(2+) transient produced by NMDA receptors is essential to normal synaptic physiology, drives the development and plasticity of excitatory central synapses, and also mediates glutamate excitotoxicity. The amplitude and time course of the Ca(2+) signal depends on the receptor's conductance and gating kinetics; these properties are themselves influenced both directly and indirectly by fluctuations in the extracellular Ca(2+) concentration. Here, we used electrophysiology and kinetic modeling to delineate the direct effects of extracellular Ca(2+) on recombinant GluN1/GluN2A receptor conductance and gating. We report that, in addition to decreasing unitary conductance, Ca(2+) also decreased channel open probability primarily by lengthening closed-channel periods. Using one-channel current recordings, we derive a kinetic model for GluN1/GluN2A receptors in physiological Ca(2+) concentrations that accurately describes macroscopic channel behaviors. This model represents a practical instrument to probe the mechanisms that control the Ca(2+) transients produced by NMDA receptors during both normal and aberrant synaptic signaling.

Inhibition of a TREK-like K+ channel current by noradrenaline requires both β1- and β2-adrenoceptors in rat atrial myocytes.

AimsNoradrenaline plays an important role in the modulation of atrial electrophysiology. However, the identity of the modulated channels, their mechanisms of modulation, and their role in the action potential remain unclear. This study aimed to investigate the noradrenergic modulation of an atrial steady-state outward current (IKss).Methods and resultsRat atrial myocyte whole-cell currents were recorded at 36°C. Noradrenaline potently inhibited IKss (IC50 = 0.90 nM, 42.1 ± 4.3% at 1 µM, n = 7) and potentiated the L-type Ca2+ current (ICaL, EC50 = 136 nM, 205 ± 40% at 1 µM, n = 6). Noradrenaline-sensitive IKss was weakly voltage-dependent, time-independent, and potentiated by the arachidonic acid analogue, 5,8,11,14-eicosatetraynoic acid (EYTA; 10 µM), or by osmotically induced membrane stretch. Noise analysis revealed a unitary conductance of 8.4 ± 0.42 pS (n = 8). The biophysical/pharmacological properties of IKss indicate a TREK-like K+ channel. The effect of noradrenaline on IKss was abolished by combined β1-/β2-adrenoceptor antagonism (1 µM propranolol or 10 µM β1-selective atenolol and 100 nM β2-selective ICI-118,551 in combination), but not by β1- or β2-antagonist alone. The action of noradrenaline could be mimicked by β2-agonists (zinterol and fenoterol) in the presence of β1-antagonist. The action of noradrenaline on IKss, but not on ICaL, was abolished by pertussis toxin (PTX) treatment. The action of noradrenaline on ICaL was mediated by β1-adrenoceptors via a PTX-insensitive pathway. Noradrenaline prolonged APD30 by 52 ± 19% (n = 5; P < 0.05), and this effect was abolished by combined β1-/β2-antagonism, but not by atenolol alone.ConclusionNoradrenaline inhibits a rat atrial TREK-like K+ channel current via a PTX-sensitive mechanism involving co-operativity of β1-/β2-adrenoceptors that contributes to atrial APD prolongation.

Tandem Pore Domain Halothane-inhibited K+ Channel Subunits THIK1 and THIK2 Assemble and Form Active Channels

Despite a high level of sequence homology, tandem pore domain halothane-inhibited K(+) channel 1 (THIK1) produces background K(+) currents, whereas THIK2 is silent. This lack of activity is due to a unique combination of intracellular retention and weak basal activity in the plasma membrane. Here, we designed THIK subunits containing dominant negative mutations (THIK1(DN) and THIK2(DN)). THIK2(DN) mutant inhibits THIK1 currents, whereas THIK1(DN) inhibits an activated form of THIK2 (THIK2-A155P-I158D). In situ proximity ligation assays and Förster/fluorescence resonance energy transfer (FRET) experiments support a physical association between THIK1 and THIK2. Next, we expressed covalent tandems of THIK proteins to obtain expression of pure heterodimers. Td-THIK1-THIK2 (where Td indicates tandem) produces K(+) currents of amplitude similar to Td-THIK1-THIK1 but with a noticeable difference in the current kinetics. Unlike Td-THIK2-THIK2 that is mainly detected in the endoplasmic reticulum, Td-THIK1-THIK2 distributes at the plasma membrane, indicating that THIK1 can mask the endoplasmic reticulum retention/retrieval motif of THIK2. Kinetics and unitary conductance of Td-THIK1-THIK2 are intermediate between THIK1 and THIK2. Altogether, these results show that THIK1 and THIK2 form active heteromeric channels, further expanding the known repertoire of K(+) channels.

Mechanosensitive Ca2 +-permeable channels in human leukemic cells: Pharmacological and molecular evidence for TRPV2

Mechanosensitive channels are present in almost every living cell, yet the evidence for their functional presence in T lymphocytes is absent. In this study, by means of the patch-clamp technique in attached and inside-out modes, we have characterized cationic channels, rapidly activated by membrane stretch in Jurkat T lymphoblasts. The half-activation was achieved at a negative pressure of ~50mm Hg. In attached mode, single channel currents displayed an inward rectification and the unitary conductance of ~40 pS at zero command voltage. In excised inside-out patches the rectification was transformed to an outward one. Mechanosensitive channels weakly discriminated between mono- and divalent cations (PCa/PNa~1) and were equally permeable for Ca2+ and Mg2+. Pharmacological analysis showed that the mechanosensitive channels were potently blocked by amiloride (1mM) and Gd3+ (10μM) in a voltage-dependent manner. They were also almost completely blocked by ruthenium red (1μM) and SKF 96365 (250μM), inhibitors of transient receptor potential vanilloid 2 (TRPV2) channels. At the same time, the channels were insensitive to 2-aminoethoxydiphenyl borate (2-APB, 100μM) or N-(p-amylcinnamoyl)anthranilic acid (ACA, 50μM), antagonists of transient receptor potential canonical (TRPC) or transient receptor potential melastatin (TRPM) channels, respectively. Human TRPV2 siRNA virtually abolished the stretch-activated current. TRPV2 are channels with multifaceted functions and regulatory mechanisms, with potentially important roles in the lymphocyte Ca2+ signaling. Implications of their regulation by mechanical stress are discussed in the context of lymphoid cells functions.

Interaction of the BKCa channel gating ring with dendrotoxins

Two classes of small homologous basic proteins, mamba snake dendrotoxins (DTX) and bovine pancreatic trypsin inhibitor (BPTI), block the large conductance Ca2+-activated K+ channel (BKCa, KCa1.1) by production of discrete subconductance events when added to the intracellular side of the membrane. This toxin-channel interaction is unlikely to be pharmacologically relevant to the action of mamba venom, but as a fortuitous ligand-protein interaction, it has certain biophysical implications for the mechanism of BKCa channel gating. In this work we examined the subconductance behavior of 9 natural dendrotoxin homologs and 6 charge neutralization mutants of δ-dendrotoxin in the context of current structural information on the intracellular gating ring domain of the BKCa channel. Calculation of an electrostatic surface map of the BKCa gating ring based on the Poisson-Boltzmann equation reveals a predominantly electronegative surface due to an abundance of solvent-accessible side chains of negatively charged amino acids. Available structure-activity information suggests that cationic DTX/BPTI molecules bind by electrostatic attraction to site(s) on the gating ring located in or near the cytoplasmic side portals where the inactivation ball peptide of the β2 subunit enters to block the channel. Such an interaction may decrease the apparent unitary conductance by altering the dynamic balance of open versus closed states of BKCa channel activation gating.

Exocytotic Machineries of Vestibular Type I and Cochlear Ribbon Synapses Display Similar Intrinsic Otoferlin-Dependent Ca2+ Sensitivity But a Different Coupling to Ca2+ Channels

The hair cell ribbon synapses of the mammalian auditory and vestibular systems differ greatly in their anatomical organization and firing properties. Notably, vestibular Type I hair cells (VHC-I) are surrounded by a single calyx-type afferent terminal that receives input from several ribbons, whereas cochlear inner hair cells (IHCs) are contacted by several individual afferent boutons, each facing a single ribbon. The specificity of the presynaptic molecular mechanisms regulating transmitter release at these different sensory ribbon synapses is not well understood. Here, we found that exocytosis during voltage activation of Ca(2+) channels displayed higher Ca(2+) sensitivity, 10 mV more negative half-maximum activation, and a smaller dynamic range in VHC-I than in IHCs. VHC-I had a larger number of Ca(2+) channels per ribbon (158 vs 110 in IHCs), but their Ca(2+) current density was twofold smaller because of a smaller open probability and unitary conductance. Using confocal and stimulated emission depletion immunofluorescence microscopy, we showed that VHC-I had fewer synaptic ribbons (7 vs 17 in IHCs) to which Cav1.3 channels are more tightly organized than in IHCs. Gradual intracellular Ca(2+) uncaging experiments revealed that exocytosis had a similar intrinsic Ca(2+) sensitivity in both VHC-I and IHCs (KD of 3.3 ± 0.6 μM and 4.0 ± 0.7 μM, respectively). In otoferlin-deficient mice, exocytosis was largely reduced in VHC-I and IHCs. We conclude that VHC-I and IHCs use a similar micromolar-sensitive otoferlin Ca(2+) sensor and that their sensory encoding specificity is essentially determined by a different functional organization of Ca(2+) channels at their synaptic ribbons.