The Journal of General Physiology recent issues

Multiple Unbiased Prospective Screens Identify TRP Channels and Their Conserved Gating Elements

Myers, B. R., Saimi, Y., Julius, D., Kung, C. — 2008-10-27

Return of the Electric Binding Site

Rothberg, B. S. — 2008-10-27

Measurements of the BKCa Channel's High-Affinity Ca2+ Binding Constants: Effects of Membrane Voltage

Sweet, T.-B., Cox, D. H. — 2008-10-27

It has been established that the large conductance Ca²⁺-activated K⁺ channel contains two types of high-affinity Ca²⁺ binding sites, termed the Ca²⁺ bowl and the RCK1 site. The affinities of these sites, and how they change as the channel opens, is still a subject of some debate. Previous estimates of these affinities have relied on fitting a series of conductance–voltage relations determined over a series of Ca²⁺ concentrations with models of channel gating that include both voltage sensing and Ca²⁺ binding. This approach requires that some model of voltage sensing be chosen, and differences in the choice of voltage-sensing model may underlie the different estimates that have been produced. Here, to better determine these affinities we have measured Ca²⁺ dose–response curves of channel activity at constant voltage for the wild-type mSlo channel (minus its low-affinity Ca²⁺ binding site) and for channels that have had one or the other Ca²⁺ binding site disabled via mutation. To accurately determine these dose–response curves we have used a series of 22 Ca²⁺ concentrations, and we have used unitary current recordings, coupled with changes in channel expression level, to measure open probability over five orders of magnitude. Our results indicate that at –80 mV the Ca²⁺ bowl has higher affinity for Ca²⁺ than does the RCK1 site in both the opened and closed conformations of the channel, and that the binding of Ca²⁺ to the RCK1 site is voltage dependent, whereas at the Ca²⁺ bowl it is not.

hERG Gating Microdomains Defined by S6 Mutagenesis and Molecular Modeling

Wynia-Smith, S. L., Gillian-Daniel, A. L., Satyshur, K. A., Robertson, G. A. — 2008-10-27

Human ether-à-go-go–related gene (hERG) channels mediate cardiac repolarization and bind drugs that can cause acquired long QT syndrome and life-threatening arrhythmias. Drugs bind in the vestibule formed by the S6 transmembrane domain, which also contains the activation gate that traps drugs in the vestibule and contributes to their efficacy of block. Although drug-binding residues have been identified, we know little about the roles of specific S6 residues in gating. We introduced cysteine mutations into the hERG channel S6 domain and measured mutational effects on the steady-state distribution and kinetics of transitions between the closed and open states. Energy-minimized molecular models based on the crystal structures of rKv1.2 (open state) and MlotiK1 and KcsA (closed state) provided structural contexts for evaluating mutant residues. The majority of mutations slowed deactivation, shifted conductance voltage curves to more negative potentials, or conferred a constitutive conductance over voltages that normally cause the channel to close. At the most intracellular extreme of the S6 region, Q664, Y667, and S668 were especially sensitive and together formed a ringed domain that occludes the pore in the closed state model. In contrast, mutation of S660, more than a full helical turn away and corresponding by alignment to a critical Shaker gate residue (V478), had little effect on gating. Multiple substitutions of chemically distinct amino acids at the adjacent V659 suggested that, upon closing, the native V659 side chain moves into a hydrophobic pocket but likely does not form the occluding gate itself. Overall, the study indicated that S6 mutagenesis disrupts the energetics primarily of channel closing and identified several residues critical for this process in the native channel.

ENaC Proteolytic Regulation by Channel-activating Protease 2

Garcia-Caballero, A., Dang, Y., He, H., Stutts, M. J. — 2008-10-27

Epithelial sodium channels (ENaCs) perform diverse physiological roles by mediating Na⁺ absorption across epithelial surfaces throughout the body. Excessive Na⁺ absorption in kidney and colon elevates blood pressure and in the airways disrupts mucociliary clearance. Potential therapies for disorders of Na⁺ absorption require better understanding of ENaC regulation. Recent work has established partial and selective proteolysis of ENaCs as an important means of channel activation. In particular, channel-activating transmembrane serine proteases (CAPs) and cognate inhibitors may be important in tissue-specific regulation of ENaCs. Although CAP2 (TMPRSS4) requires catalytic activity to activate ENaCs, there is not yet evidence of ENaC fragments produced by this serine protease and/or identification of the site(s) where CAP2 cleaves ENaCs. Here, we report that CAP2 cleaves at multiple sites in all three ENaC subunits, including cleavage at a conserved basic residue located in the vicinity of the degenerin site (-K561, β-R503, and -R515). Sites in -ENaC at K149/R164/K169/R177 and furin-consensus sites in -ENaC (R205/R231) and -ENaC (R138) are responsible for ENaC fragments observed in oocytes coexpressing CAP2. However, the only one of these demonstrated cleavage events that is relevant for the channel activation by CAP2 takes place in -ENaC at position R138, the previously identified furin-consensus cleavage site. Replacement of arginine by alanine or glutamine (,β,R138A/Q) completely abolished both the Na⁺ current (I_Na) and a 75-kD -ENaC fragment at the cell surface stimulated by CAP2. Replacement of -ENaC R138 with a conserved basic residue, lysine, preserved both the CAP2-induced I_Na and the 75-kD -ENaC fragment. These data strongly support a model where CAP2 activates ENaCs by cleaving at R138 in -ENaC.

Rescue of Volume-regulated Anion Current by Bestrophin Mutants with Altered Charge Selectivity

Chien, L.-T., Hartzell, H. C. — 2008-10-27

Mutations in human bestrophin-1 are linked to various kinds of retinal degeneration. Although it has been proposed that bestrophins are Ca²⁺-activated Cl^– channels, definitive proof is lacking partly because mice with the bestrophin-1 gene deleted have normal Ca²⁺-activated Cl^– currents. Here, we provide compelling evidence to support the idea that bestrophin-1 is the pore-forming subunit of a cell volume-regulated anion channel (VRAC) in Drosophila S2 cells. VRAC was abolished by treatment with RNAi to Drosophila bestrophin-1. VRAC was rescued by overexpressing bestrophin-1 mutants with altered biophysical properties and responsiveness to sulfhydryl reagents. In particular, the ionic selectivity of the F81C mutant changed from anionic to cationic when the channel was treated with the sulfhydryl reagent, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES^–) (P_Cs/P_Cl = 0.25 for native and 2.38 for F81C). The F81E mutant was 1.3 times more permeable to Cs⁺ than Cl^–. The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit. F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES^– treatment, although the effect is quantitatively smaller than in S2 cells. To test whether bestrophins are VRACs in mammalian cells, we compared VRACs in peritoneal macrophages from wild-type mice and mice with both bestrophin-1 and bestrophin-2 disrupted (best1^–/–/best2^–/–). VRACs were identical in wild-type and best1^–/–/best2^–/– mice, showing that bestrophins are unlikely to be the classical VRAC in mammalian cells.

Properties of the Inner Pore Region of TRPV1 Channels Revealed by Block with Quaternary Ammoniums

Jara-Oseguera, A., Llorente, I., Rosenbaum, T., Islas, L. D. — 2008-10-27

The transient receptor potential vanilloid 1 (TRPV1) nonselective cationic channel is a polymodal receptor that activates in response to a wide variety of stimuli. To date, little structural information about this channel is available. Here, we used quaternary ammonium ions (QAs) of different sizes in an effort to gain some insight into the nature and dimensions of the pore of TRPV1. We found that all four QAs used, tetraethylammonium (TEA), tetrapropylammonium (TPrA), tetrabutylammonium, and tetrapentylammonium, block the TRPV1 channel from the intracellular face of the channel in a voltage-dependent manner, and that block by these molecules occurs with different kinetics, with the bigger molecules becoming slower blockers. We also found that TPrA and the larger QAs can only block the channel in the open state, and that they interfere with the channel's activation gate upon closing, which is observed as a slowing of tail current kinetics. TEA does not interfere with the activation gate, indicating that this molecule can reside in its blocking site even when the channel is closed. The dependence of the rate constants on the size of the blocker suggests a size of around 10 Å for the inner pore of TRPV1 channels.

The P2X7 Receptor Channel Pore Dilates under Physiological Ion Conditions

Yan, Z., Li, S., Liang, Z., Tomic, M., Stojilkovic, S. S. — 2008-10-27

Activation of the purinergic P2X₇ receptor leads to the rapid opening of an integral ion channel that is permeable to small cations. This is followed by a gradual increase in permeability to fluorescent dyes by integrating the actions of the pannexin-1 channel. Here, we show that during the prolonged agonist application a rapid current that peaked within 200 ms was accompanied with a slower current that required tens of seconds to reach its peak. The secondary rise in current was observed under different ionic conditions and temporally coincided with the development of conductivity to larger organic cations. The biphasic response was also observed in cells with blocked pannexin channels and in cells not expressing these channels endogenously. The biphasic current was preserved in N-terminal T15A, T15S, and T15V mutants that have low or no permeability to organic cations, reflecting enhanced permeability to inorganic cations. In contrast, the T15E, T15K, and T15W mutants, and the 18 mutant with deleted P2X₇ receptor–specific 18–amino acid C-terminal segment, were instantaneously permeable to organic cations and generated high amplitude monophasic currents. These results indicate that the P2X₇ receptor channel dilates under physiological ion conditions, leading to generation of biphasic current, and that this process is controlled by residues near the intracellular side of the channel pore.

Contribution of the Myosin Binding Protein C Motif to Functional Effects in Permeabilized Rat Trabeculae

Razumova, M. V., Bezold, K. L., Tu, A.-Y., Regnier, M., Harris, S. P. — 2008-10-27

Myosin binding protein C (MyBP-C) is a thick-filament protein that limits cross-bridge cycling rates and reduces myocyte power output. To investigate mechanisms by which MyBP-C affects contraction, we assessed effects of recombinant N-terminal domains of cardiac MyBP-C (cMyBP-C) on contractile properties of permeabilized rat cardiac trabeculae. Here, we show that N-terminal fragments of cMyBP-C that contained the first three immunoglobulin domains of cMyBP-C (i.e., C0, C1, and C2) plus the unique linker sequence termed the MyBP-C "motif" or "m-domain" increased Ca²⁺ sensitivity of tension and increased rates of tension redevelopment (i.e., k_tr) at submaximal levels of Ca²⁺. At concentrations ≥20 µM, recombinant proteins also activated force in the absence of Ca²⁺ and inhibited maximum Ca²⁺-activated force. Recombinant proteins that lacked the combination of C1 and the motif did not affect contractile properties. These results suggest that the C1 domain plus the motif constitute a functional unit of MyBP-C that can activate the thin filament.

Kinetics of Turn-offs of Frog Rod Phototransduction Cascade

Astakhova, L. A., Firsov, M. L., Govardovskii, V. I. — 2008-10-27

The time course of the light-induced activity of phototrandsuction effector enzyme cGMP-phosphodiesterase (PDE) is shaped by kinetics of rhodopsin and transducin shut-offs. The two processes are among the key factors that set the speed and sensitivity of the photoresponse and whose regulation contributes to light adaptation. The aim of this study was to determine time courses of flash-induced PDE activity in frog rods that were dark adapted or subjected to nonsaturating steady background illumination. PDE activity was computed from the responses recorded from solitary rods with the suction pipette technique in Ca²⁺-clamping solution.

A flash applied in the dark-adapted state elicits a wave of PDE activity whose rising and decaying phases have characteristic times near 0.5 and 2 seconds, respectively. Nonsaturating steady background shortens both phases roughly to the same extent. The acceleration may exceed fivefold at the backgrounds that suppress 70% of the dark current.

The time constant of the process that controls the recovery from super-saturating flashes (so-called dominant time constant) is adaptation independent and, hence, cannot be attributed to either of the processes that shape the main part of the PDE wave. We hypothesize that the dominant time constant in frog rods characterizes arrestin binding to rhodopsin partially inactivated by phosphorylation. A mathematical model of the cascade that considers two-stage rhodopsin quenching and transducin inactivation can mimic experimental PDE activity quite well. The effect of light adaptation on the PDE kinetics can be reproduced in the model by concomitant acceleration on both rhodopsin phosphorylation and transducin turn-off, but not by accelerated arrestin binding. This suggests that not only rhodopsin but also transducin shut-off is under adaptation control.

The mouth of a dense-core vesicle opens and closes in a concerted action regulated by calcium and amphiphysin

Llobet, A., Wu, M., Lagnado, L. — 2008-09-29

Excitation-Contraction Coupling of the Mouse Embryonic Cardiomyocyte

Rapila, R., Korhonen, T., Tavi, P. — 2008-09-29

In the mammalian embryo, the primitive tubular heart starts beating during the first trimester of gestation. These early heartbeats originate from calcium-induced contractions of the developing heart muscle cells. To explain the initiation of this activity, two ideas have been presented. One hypothesis supports the role of spontaneously activated voltage-gated calcium channels, whereas the other emphasizes the role of Ca²⁺ release from intracellular stores initiating spontaneous intracellular calcium oscillations. We show with experiments that both of these mechanisms coexist and operate in mouse cardiomyocytes during embryonic days 9–11. Further, we characterize how inositol-3-phosphate receptors regulate the frequency of the sarcoplasmic reticulum calcium oscillations and thus the heartbeats. This study provides a novel view of the regulation of embryonic cardiomyocyte activity, explaining the functional versatility of developing cardiomyocytes and the origin and regulation of the embryonic heartbeat.

Mathematical Model of Mouse Embryonic Cardiomyocyte Excitation-Contraction Coupling

Korhonen, T., Rapila, R., Tavi, P. — 2008-09-29

Excitation–contraction (E–C) coupling is the mechanism that connects the electrical excitation with cardiomyocyte contraction. Embryonic cardiomyocytes are not only capable of generating action potential (AP)-induced Ca²⁺ signals and contractions (E–C coupling), but they also can induce spontaneous pacemaking activity. The spontaneous activity originates from spontaneous Ca²⁺ releases from the sarcoplasmic reticulum (SR), which trigger APs via the Na⁺/Ca²⁺ exchanger (NCX). In the AP-driven mode, an external stimulus triggers an AP and activates voltage-activated Ca²⁺ intrusion to the cell. These complex and unique features of the embryonic cardiomyocyte pacemaking and E–C coupling have never been assessed with mathematical modeling. Here, we suggest a novel mathematical model explaining how both of these mechanisms can coexist in the same embryonic cardiomyocytes. In addition to experimentally characterized ion currents, the model includes novel heterogeneous cytosolic Ca²⁺ dynamics and oscillatory SR Ca²⁺ handling. The model reproduces faithfully the experimentally observed fundamental features of both E–C coupling and pacemaking. We further validate our model by simulating the effect of genetic modifications on the hyperpolarization-activated current, NCX, and the SR Ca²⁺ buffer protein calreticulin. In these simulations, the model produces a similar functional alteration to that observed previously in the genetically engineered mice, and thus provides mechanistic explanations for the cardiac phenotypes of these animals. In general, this study presents the first model explaining the underlying cellular mechanism for the origin and the regulation of the heartbeat in early embryonic cardiomyocytes.

ATP Inhibition of CLC-1 Is Controlled by Oxidation and Reduction

Zhang, X.-D., Tseng, P.-Y., Chen, T.-Y. — 2008-09-29

The effect of intracellular adenosine triphosphate (ATP) on the "common gating" of the CLC-1 chloride channel has been studied by several laboratories with controversial results. Our previous study on the channel expressed in Xenopus oocytes using excised inside-out patch-clamp methods showed a robust effect of ATP in shifting the open probability curve of the common gate toward more depolarizing voltages (Tseng, P.Y., B. Bennetts, and T.Y. Chen. 2007. J. Gen. Physiol. 130:217–221). The results were consistent with those from studying the channel expressed in mammalian cells using whole cell recording methods (Bennetts, B., M.W. Parker, and B.A. Cromer. 2007. J. Biol. Chem. 282:32780–32791). However, a recent study using excised-patch recording methods for channels expressed in Xenopus oocytes reported that ATP had no direct effect on CLC-1 (Zifarelli, G., and M. Pusch. 2008. J. Gen. Physiol. 131:109–116). Here, we report that oxidation of CLC-1 may be the culprit underlying the controversy. When patches were excised from mammalian cells, the sensitivity to ATP was lost quickly—within 2–3 min. This loss of ATP sensitivity could be prevented or reversed by reducing agents. On the other hand, CLC-1 expressed in Xenopus oocytes lost the ATP sensitivity when patches were treated with oxidizing reagents. These results suggest a novel view in muscle physiology that the mechanisms controlling muscle fatigability may include the oxidation of CLC-1.

Luminal Mg2+, A Key Factor Controlling RYR2-mediated Ca2+ Release: Cytoplasmic and Luminal Regulation Modeled in a Tetrameric Channel

Laver, D. R., Honen, B. N. — 2008-09-29

In cardiac muscle, intracellular Ca²⁺ and Mg²⁺ are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [Ca²⁺] in the SR ([Ca²⁺]_L) stimulates the Ca²⁺ release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal Mg²⁺, which has not been regarded as an important regulator of Ca²⁺ release.

The effects of luminal Ca²⁺ and Mg²⁺ on sheep RYR2 were measured in lipid bilayers. Cytoplasmic and luminal Ca²⁺ produced a synergistic increase in the opening rate of RYRs. A novel, high affinity inhibition of RYR2 by luminal Mg²⁺ was observed, pointing to an important physiological role for luminal Mg²⁺ in cardiac muscle. At diastolic [Ca²⁺]_C, luminal Mg²⁺ inhibition was voltage independent, with K_i = 45 µM at luminal [Ca²⁺] ([Ca²⁺]_L) = 100 µM. Luminal and cytoplasmic Mg²⁺ inhibition was alleviated by increasing [Ca²⁺]_L or [Ca²⁺]_C. Ca²⁺ and Mg²⁺ on opposite sides of the bilayer exhibited competitive effects on RYRs, indicating that they can compete via the pore for common sites.

The data were accurately fitted by a model based on a tetrameric RYR structure with four Ca²⁺-sensing mechanisms on each subunit: activating luminal L-site (40-µM affinity for Mg²⁺ and Ca²⁺), cytoplasmic A-site (1.2 µM for Ca²⁺ and 60 µM for Mg²⁺), inactivating cytoplasmic I₁-site (~10 mM for Ca²⁺ and Mg²⁺), and I₂-site (1.2 µM for Ca²⁺). Activation of three or more subunits will cause channel opening. Mg²⁺ inhibition occurs primarily by Mg²⁺ displacing Ca²⁺ from the L- and A-sites, and Mg²⁺ fails to open the channel.

The model predicts that under physiological conditions, SR load–dependent Ca²⁺ release (1) is mainly determined by Ca²⁺ displacement of Mg²⁺ from the L-site as SR loading increases, and (2) depends on the properties of both luminal and cytoplasmic activation mechanisms.

Gating Pore Currents in DIIS4 Mutations of NaV1.4 Associated with Periodic Paralysis: Saturation of Ion Flux and Implications for Disease Pathogenesis

Struyk, A. F., Markin, V. S., Francis, D., Cannon, S. C. — 2008-09-29

S4 voltage–sensor mutations in CaV1.1 and NaV1.4 channels cause the human muscle disorder hypokalemic periodic paralysis (HypoPP). The mechanism whereby these mutations predispose affected sarcolemma to attacks of sustained depolarization and loss of excitability is poorly understood. Recently, three HypoPP mutations in the domain II S4 segment of NaV1.4 were shown to create accessory ionic permeation pathways, presumably extending through the aqueous gating pore in which the S4 segment resides. However, there are several disparities between reported gating pore currents from different investigators, including differences in ionic selectivity and estimates of current amplitude, which in turn have important implications for the pathological relevance of these aberrant currents. To clarify the features of gating pore currents arising from different DIIS4 mutants, we recorded gating pore currents created by HypoPP missense mutations at position R666 in the rat isoform of Nav1.4 (the second arginine from the outside, at R672 in human NaV1.4). Extensive measurements were made for the index mutation, R666G, which created a gating pore that was permeable to K⁺ and Na⁺. This current had a markedly shallow slope conductance at hyperpolarized voltages and robust inward rectification, even when the ionic gradient strongly favored outward ionic flow. These characteristics were accounted for by a barrier model incorporating a voltage-gated permeation pathway with a single cation binding site oriented near the external surface of the electrical field. The amplitude of the R666G gating pore current was similar to the amplitude of a previously described proton-selective current flowing through the gating pore in rNaV1.4-R663H mutant channels. Currents with similar amplitude and cation selectivity were also observed in R666S and R666C mutant channels, while a proton-selective current was observed in R666H mutant channels. These results add support to the notion that HypoPP mutations share a common biophysical profile comprised of a low-amplitude inward current at the resting potential that may contribute to the pathological depolarization during attacks of weakness.

Steady-state Function of the Ubiquitous Mammalian Na/H Exchanger (NHE1) in Relation to Dimer Coupling Models with 2Na/2H Stoichiometry

Fuster, D., Moe, O. W., Hilgemann, D. W. — 2008-09-29

We describe the steady-state function of the ubiquitous mammalian Na/H exchanger (NHE)1 isoform in voltage-clamped Chinese hamster ovary cells, as well as other cells, using oscillating pH-sensitive microelectrodes to quantify proton fluxes via extracellular pH gradients. Giant excised patches could not be used as gigaseal formation disrupts NHE activity within the patch. We first analyzed forward transport at an extracellular pH of 8.2 with no cytoplasmic Na (i.e., nearly zero-trans). The extracellular Na concentration dependence is sigmoidal at a cytoplasmic pH of 6.8 with a Hill coefficient of 1.8. In contrast, at a cytoplasmic pH of 6.0, the Hill coefficient is <1, and Na dependence often appears biphasic. Results are similar for mouse skin fibroblasts and for an opossum kidney cell line that expresses the NHE3 isoform, whereas NHE1^–/– skin fibroblasts generate no proton fluxes in equivalent experiments. As proton flux is decreased by increasing cytoplasmic pH, the half-maximal concentration (K_1/2) of extracellular Na decreases less than expected for simple consecutive ion exchange models. The K_1/2 for cytoplasmic protons decreases with increasing extracellular Na, opposite to predictions of consecutive exchange models. For reverse transport, which is robust at a cytoplasmic pH of 7.6, the K_1/2 for extracellular protons decreases only a factor of 0.4 when maximal activity is decreased fivefold by reducing cytoplasmic Na. With 140 mM of extracellular Na and no cytoplasmic Na, the K_1/2 for cytoplasmic protons is 50 nM (pH 7.3; Hill coefficient, 1.5), and activity decreases only 25% with extracellular acidification from 8.5 to 7.2. Most data can be reconstructed with two very different coupled dimer models. In one model, monomers operate independently at low cytoplasmic pH but couple to translocate two ions in "parallel" at alkaline pH. In the second "serial" model, each monomer transports two ions, and translocation by one monomer allosterically promotes translocation by the paired monomer in opposite direction. We conclude that a large fraction of mammalian Na/H activity may occur with a 2Na/2H stoichiometry.

TRPP2 and TRPV4 form a polymodal sensory channel complex

2008-08-25

Divalent Cations Regulate Connexin Hemichannels by Modulating Intrinsic Voltage-dependent Gating

Verselis, V. K., Srinivas, M. — 2008-08-25

Connexin hemichannels are robustly regulated by voltage and divalent cations. The basis of voltage-dependent gating, however, has been questioned with reports that it is not intrinsic to hemichannels, but rather is derived from divalent cations acting as gating particles that block the pore in a voltage-dependent manner. Previously, we showed that connexin hemichannels possess two types of voltage-dependent gating, termed V_j and loop gating, that in Cx46 operate at opposite voltage polarities, positive and negative, respectively. Using recordings of single Cx46 hemichannels, we found both forms of gating persist in solutions containing no added Mg²⁺ and EGTA to chelate Ca²⁺. Although loop gating persists, it is significantly modulated by changing levels of extracellular divalent cations. When extracellular divalent cation concentrations are low, large hyperpolarizing voltages, exceeding –100 mV, could still drive Cx46 hemichannels toward closure. However, gating is characterized by continuous flickering of the unitary current interrupted by occasional, brief sojourns to a quiet closed state. Addition of extracellular divalent cations, in this case Mg²⁺, results in long-lived residence in a quiet closed state, suggesting that hyperpolarization drives the hemichannel to close, perhaps by initiating movements in the extracellular loops, and that divalent cations stabilize the fully closed conformation. Using excised patches, we found that divalent cations are only effective from the extracellular side, indicative that the binding site is not cytoplasmic or in the pore, but rather extracellular. V_j gating remains essentially unaffected by changing levels of extracellular divalent cations. Thus, we demonstrate that both forms of voltage dependence are intrinsic gating mechanisms in Cx46 hemichannels and that the action of external divalent cations is to selectively modulate loop gating.

Glucose and GLP-1 Stimulate cAMP Production via Distinct Adenylyl Cyclases in INS-1E Insulinoma Cells

Ramos, L. S., Zippin, J. H., Kamenetsky, M., Buck, J., Levin, L. R. — 2008-08-25

In β cells, both glucose and hormones, such as GLP-1, stimulate production of the second messenger cAMP, but glucose and GLP-1 elicit distinct cellular responses. We now show in INS-1E insulinoma cells that glucose and GLP-1 produce cAMP with distinct kinetics via different adenylyl cyclases. GLP-1 induces a rapid cAMP signal mediated by G protein–responsive transmembrane adenylyl cyclases (tmAC). In contrast, glucose elicits a delayed cAMP rise mediated by bicarbonate, calcium, and ATP-sensitive soluble adenylyl cyclase (sAC). This glucose-induced, sAC-dependent cAMP rise is dependent upon calcium influx and is responsible for the glucose-induced activation of the mitogen-activated protein kinase (ERK1/2) pathway. These results demonstrate that sAC-generated and tmAC-generated cAMP define distinct signaling cascades.

Nitric Oxide-mediated Modulation of Synaptic Activity by Astrocytic P2Y Receptors

Mehta, B., Begum, G., Joshi, N. B., Joshi, P. G. — 2008-08-25

We investigated the mechanism of synaptic suppression by P2Y receptors in mixed hippocampal cultures wherein networked neurons exhibit synchronized Ca²⁺ oscillations (SCO) due to spontaneous glutamatergic synaptic transmission. Pharmacological studies suggested that SCO suppression was mediated by P2Y2/P2Y4 receptors. Immunostaining studies and characterization of ATP/UTP-stimulated Ca²⁺ responses in solitary neurons and astrocytes revealed that the SCO attenuation was effectuated by astrocytes. We demonstrate that nitric oxide released from activated astrocytes causes synaptic suppression by inhibiting neurotransmitter release. Physiological concentrations of ATP and UTP evoked NO production in astrocytes. SCO suppression was considerably diminished by removal of extracellular NO by membrane-impermeable scavenger c-PTIO or by pretreatment of cells with nitric oxide synthase inhibitor L-NAME. The nitric oxide donor DETA/NO effectively suppressed the SCO. ATP/UTP inhibited KCl-induced exocytosis at presynaptic terminals in an NO-dependent manner. In the absence of exogenously added ATP/UTP, both the NO scavenger and NOS inhibitor enhanced the frequency of SCO, implying that astrocytes release NO during spontaneous synaptic activity and exert a suppressive effect. We report for the first time that under physiological conditions astrocytes use NO as a messenger molecule to modulate the synaptic strength in the networked neurons.

Preventing Voltage-dependent Gating of Anthrax Toxin Channels Using Engineered Disulfides

Anderson, D. S., Blaustein, R. O. — 2008-08-25

The channel-forming component of anthrax toxin, (PA₆₃)₇, is a heptameric water-soluble protein at neutral pH, but under acidic conditions it spontaneously inserts into lipid bilayers to form a 14-stranded β-barrel ion-conducting channel. This channel plays a vital role in anthrax pathogenesis because it serves as a conduit for the membrane translocation of the two enzymatic components of anthrax toxin, lethal factor and edema factor. Anthrax channels open and close in response to changes in transmembrane voltage, a property shared by several other pore-forming toxins. We have discovered an unexpected phenomenon in cysteine-substituted channels that provides a window into this gating process: their normal voltage-dependent gating can be abolished by reaction with methanethiosulfonate (MTS) reagents or exposure to oxidizing conditions. Remarkably, this perturbation is seen with cysteines substituted at sites all along the ~100 Å length of the channel's β-barrel. In contrast, reaction with N-ethylmaleimide, a thiol-reactive compound that does not form a mixed disulfide, does not affect gating at any of the sites tested. These findings, coupled with our biochemical detection of dimers, have led us to conclude that MTS reagents are catalyzing the formation of intersubunit disulfide bonds that lock channels in a conducting state, and that voltage gating requires a conformational change that involves the entire β-barrel.

A Carboxy-terminal Inter-Helix Linker As the Site of Phosphatidylinositol 4,5-Bisphosphate Action on Kv7 (M-type) K+ Channels

Hernandez, C. C., Zaika, O., Shapiro, M. S. — 2008-08-25

The regulation of M-type (KCNQ [Kv7]) K⁺ channels by phosphatidylinositol 4,5-bisphosphate (PIP₂) has perhaps the best correspondence to physiological signaling, but the site of action and structural motif of PIP₂ on these channels have not been established. Using single-channel recordings of chimeras of Kv7.3 and 7.4 channels with highly differential PIP₂ sensitivities, we localized a carboxy-terminal inter-helix linker as the primary site of PIP₂ action. Point mutants within this linker in Kv7.2 and Kv7.3 identified a conserved cluster of basic residues that interact with the lipid using electrostatic and hydrogen bonds. Homology modeling of this putative PIP₂-binding linker in Kv7.2 and Kv7.3 using the solved structure of Kir2.1 and Kir3.1 channels as templates predicts a structure of Kv7.2 and 7.3 very similar to the Kir channels, and to the seven-β-sheet barrel motif common to other PIP₂-binding domains. Phosphoinositide-docking simulations predict affinities and interaction energies in accord with the experimental data, and furthermore indicate that the precise identity of residues in the interacting pocket alter channel–PIP₂ interactions not only by altering electrostatic energies, but also by allosterically shifting the structure of the lipid-binding surface. The results are likely to shed light on the general structural mechanisms of phosphoinositide regulation of ion channels.

Mutations of Nonconserved Residues within the Calcium Channel {alpha}1-interaction Domain Inhibit {beta}-Subunit Potentiation

Gonzalez-Gutierrez, G., Miranda-Laferte, E., Naranjo, D., Hidalgo, P., Neely, A. — 2008-08-25

Voltage-dependent calcium channels consist of a pore-forming subunit (Ca_V₁) that includes all the molecular determinants of a voltage-gated channel, and several accessory subunits. The ancillary β-subunit (Ca_Vβ) is a potent activator of voltage-dependent calcium channels, but the mechanisms and structural bases of this regulation remain elusive. Ca_Vβ binds reversibly to a conserved consensus sequence in Ca_V₁, the ₁-interaction domain (AID), which forms an -helix when complexed with Ca_Vβ. Conserved aromatic residues face to one side of the helix and strongly interact with a hydrophobic pocket on Ca_Vβ. Here, we studied the effect of mutating residues located opposite to the AID-Ca_Vβ contact surface in Ca_V1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other Ca_V₁ subunits (E462R, K465N, D469S, and Q473K) hinders Ca_Vβ's ability to increase ionic-current to charge-movement ratio (I/Q) without changing the apparent affinity for Ca_Vβ. At the single channel level, these Ca_V1.2 mutants coexpressed with Ca_Vβ_2a visit high open probability mode less frequently than wild-type channels. On the other hand, Ca_V1.2 carrying either a mutation in the conserved tryptophan residue (W470S, which impairs Ca_Vβ binding), or a deletion of the whole AID sequence, does not exhibit Ca_Vβ-induced increase in I/Q. In addition, we observed a shift in the voltage dependence of activation by +12 mV in the AID-deleted channel in the absence of Ca_Vβ, suggesting a direct participation of these residues in the modulation of channel activation. Our results show that Ca_Vβ-dependent potentiation arises primarily from changes in the modal gating behavior. We envision that Ca_Vβ spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by Ca_Vβ.

SERCA pump activity is physiologically regulated by presenilin and regulates amyloid {beta} production

2008-07-28

Nav1.4 Deregulation in Dystrophic Skeletal Muscle Leads to Na+ Overload and Enhanced Cell Death

Hirn, C., Shapovalov, G., Petermann, O., Roulet, E., Ruegg, U. T. — 2008-07-28

Duchenne muscular dystrophy (DMD) is a hereditary degenerative disease manifested by the absence of dystrophin, a structural, cytoskeletal protein, leading to muscle degeneration and early death through respiratory and cardiac muscle failure. Whereas the rise of cytosolic Ca²⁺ concentrations in muscles of mdx mouse, an animal model of DMD, has been extensively documented, little is known about the mechanisms causing alterations in Na⁺ concentrations. Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Na_v1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na⁺ concentrations found in muscle from mdx mice. The absence of dystrophin modifies the expression level and gating properties of Na_v1.4, leading to an increased Na⁺ concentration under the sarcolemma. Moreover, the distribution of Na_v1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin -1, thus suggesting that syntrophin is an important linker between dystrophin and Na_v1.4. Additionally, we show that these modifications of Na_v1.4 gating properties and increased Na⁺ concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na⁺ overload can be reversed by 3 nM tetrodotoxin, a specific Na_v1.4 blocker.

Voltage Clamp Fluorimetry Reveals a Novel Outer Pore Instability in a Mammalian Voltage-gated Potassium Channel

Vaid, M., Claydon, T. W., Rezazadeh, S., Fedida, D. — 2008-07-28

Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K⁺ ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3–S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 ± 3% of the total signal and occurred with a of 3.4 ± 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K⁺ to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.

Permeation and Gating in CaV3.1 ({alpha}1G) T-type Calcium Channels Effects of Ca2+, Ba2+, Mg2+, and Na+

Khan, N., Gray, I. P., Obejero-Paz, C. A., Jones, S. W. — 2008-07-28

We examined the concentration dependence of currents through Ca_V3.1 T-type calcium channels, varying Ca²⁺ and Ba²⁺ over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current–voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by P_Ca/P_Na = 87 and P_Ca/P_Ba = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent K_d values were similar for Ca²⁺ and Ba²⁺, both for block of currents carried by Na⁺ (3 µM for Ca²⁺ vs. 4 µM for Ba²⁺, at –30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca²⁺ vs. 2.5 mM for Ba²⁺; nearly voltage independent). Block by 3–10 µM Ca²⁺ was time dependent, described by bimolecular kinetics with binding at ~3 x 10⁸ M^–1s^–1 and voltage-dependent exit. Ca²⁺_o, Ba²⁺_o, and Mg²⁺_o also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e^– per 98 Å² from Gouy-Chapman theory. Additionally, inward currents inactivated ~35% faster in Ba²⁺_o (vs. Ca²⁺_o or Na⁺_o). The accelerated inactivation in Ba²⁺_o correlated with the transition from Na⁺ to Ba²⁺ permeation, suggesting that Ba²⁺_o speeds inactivation by occupying the pore. We conclude that the selectivity of the "surface charge" among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca²⁺ or Ba²⁺.

Ni2+ Block of CaV3.1 ({alpha}1G) T-type Calcium Channels

Obejero-Paz, C. A., Gray, I. P., Jones, S. W. — 2008-07-28

Ni²⁺ inhibits current through calcium channels, in part by blocking the pore, but Ni²⁺ may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of Ni²⁺ on Ca_V3.1 is affected by permeant ions. We find two components to block by Ni²⁺, a rapid block with little voltage dependence, and a slow block most visible as accelerated tail currents. Rapid block is weaker for outward vs. inward currents (apparent K_d = 3 vs. 1 mM Ni²⁺, with 2 mM Ca²⁺ or Ba²⁺) and is reduced at high permeant ion concentration (110 vs. 2 mM Ca²⁺ or Ba²⁺). Slow block depends both on the concentration and on the identity of the permeant ion (Ca²⁺ vs. Ba²⁺ vs. Na⁺). Slow block is 2–3x faster in Ba²⁺ than in Ca²⁺ (2 or 110 mM), and is ~10x faster with 2 vs. 110 mM Ca²⁺ or Ba²⁺. Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations (~3 µM Ca²⁺). We conclude that both fast and slow block of Ca_V3.1 by Ni²⁺ are most consistent with occlusion of the pore. The exit rate of Ni²⁺ for slow block is reduced at high Ni²⁺ concentrations, suggesting that the site responsible for fast block can "lock in" slow block by Ni²⁺, at a site located deeper within the pore. In contrast to the complex pore block observed for Ca_V3.1, inhibition of Ca_V3.2 by Ni²⁺ was essentially independent of voltage, and was similar in 2 mM Ca²⁺ vs. Ba²⁺, consistent with inhibition by a different mechanism, at a site outside the pore.

{alpha}-Scorpion Toxin Impairs a Conformational Change that Leads to Fast Inactivation of Muscle Sodium Channels

Campos, F. V., Chanda, B., Beirao, P. S.L., Bezanilla, F. — 2008-07-28

-Scorpion toxins bind in a voltage-dependent way to site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. We have used Ts3, an -scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared with control conditions. Ts3 accelerated the gating current kinetics, decreasing the contribution of the slow component to the ON gating current decay, indicating that S4-DIV was specifically inhibited by the toxin. In addition, Ts3 accelerated and decreased the fraction of charge in the slow component of the OFF gating current decay, which reflects an acceleration in the recovery from the fast inactivation. Site-specific fluorescence measurements indicate that Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. We also measured the fluorescent signals produced by the movement of the first three voltage sensors to test whether the bound Ts3 affects the movement of the other voltage sensors. While the fluorescence–voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.

Nicotinic Receptor Interloop Proline Anchors {beta}1-{beta}2 and Cys loops in Coupling Agonist Binding to Channel Gating

Lee, W. Y., Free, C. R., Sine, S. M. — 2008-07-28

Nicotinic acetylcholine receptors (AChRs) mediate rapid excitatory synaptic transmission throughout the peripheral and central nervous systems. They transduce binding of nerve-released ACh into opening of an intrinsic channel, yet the structural basis underlying transduction is not fully understood. Previous studies revealed a principal transduction pathway in which Arg 209 of the pre-M1 domain and Glu 45 of the β1–β2 loop functionally link the two regions, positioning Val 46 of the β1–β2 loop in a cavity formed by Pro 272 through Ser 269 of the M2–M3 loop. Here we investigate contributions of residues within and proximal to this pathway using single-channel kinetic analysis, site-directed mutagenesis, and thermodynamic mutant cycle analysis. We find that in contributing to channel gating, Val 46 and Val 132 of the signature Cys loop couple energetically to Pro 272. Furthermore, these residues are optimized in both their size and hydrophobicity to mediate rapid and efficient channel gating, suggesting naturally occurring substitutions at these positions enable a diverse range of gating rate constants among the Cys-loop receptor superfamily. The overall results indicate that Pro 272 functionally couples to flanking Val residues extending from the β1–β2 and Cys loops within the ACh binding to channel opening transduction pathway.

ACTH Inhibits bTREK-1 K+ Channels through Multiple cAMP-dependent Signaling Pathways

Liu, H., Enyeart, J. A., Enyeart, J. J. — 2008-07-28

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K⁺ channels that set the resting membrane potential and function pivotally in the physiology of cortisol secretion. Inhibition of these K⁺ channels by adrenocorticotropic hormone (ACTH) or cAMP is coupled to depolarization and Ca²⁺ entry. The mechanism of ACTH and cAMP-mediated inhibition of bTREK-1 was explored in whole cell patch clamp recordings from AZF cells. Inhibition of bTREK-1 by ACTH and forskolin was not affected by the addition of both H-89 and PKI(6–22) amide to the pipette solution at concentrations that completely blocked activation of cAMP-dependent protein kinase (PKA) in these cells. The ACTH derivative, O-nitrophenyl, sulfenyl-adrenocorticotropin (NPS-ACTH), at concentrations that produced little or no activation of PKA, inhibited bTREK-1 by a Ca²⁺-independent mechanism. Northern blot analysis showed that bovine AZF cells robustly express mRNA for Epac2, a guanine nucleotide exchange protein activated by cAMP. The selective Epac activator, 8-pCPT-2'-O-Me-cAMP, applied intracellularly through the patch pipette, inhibited bTREK-1 (IC₅₀ = 0.63 µM) at concentrations that did not activate PKA. Inhibition by this agent was unaffected by PKA inhibitors, including RpcAMPS, but was eliminated in the absence of hydrolyzable ATP. Culturing AZF cells in the presence of ACTH markedly reduced the expression of Epac2 mRNA. 8-pCPT-2'-O-Me-cAMP failed to inhibit bTREK-1 current in AZF cells that had been treated with ACTH for 3–4 d while inhibition by 8-br-cAMP was not affected. 8-pCPT-2'-O-Me-cAMP failed to inhibit bTREK-1 expressed in HEK293 cells, which express little or no Epac2. These findings demonstrate that, in addition to the well-described PKA-dependent TREK-1 inhibition, ACTH, NPS-ACTH, forskolin, and 8-pCPT-2'-O-Me-cAMP also inhibit these K⁺ channels by a PKA-independent signaling pathway. The convergent inhibition of bTREK-1 through parallel PKA- and Epac-dependent mechanisms may provide for failsafe membrane depolarization by ACTH.

Linking Exponential Components to Kinetic States in Markov Models for Single-Channel Gating

Shelley, C., Magleby, K. L. — 2008-07-28

Discrete state Markov models have proven useful for describing the gating of single ion channels. Such models predict that the dwell-time distributions of open and closed interval durations are described by mixtures of exponential components, with the number of exponential components equal to the number of states in the kinetic gating mechanism. Although the exponential components are readily calculated (Colquhoun and Hawkes, 1982, Phil. Trans. R. Soc. Lond. B. 300:1–59), there is little practical understanding of the relationship between components and states, as every rate constant in the gating mechanism contributes to each exponential component. We now resolve this problem for simple models. As a tutorial we first illustrate how the dwell-time distribution of all closed intervals arises from the sum of constituent distributions, each arising from a specific gating sequence. The contribution of constituent distributions to the exponential components is then determined, giving the relationship between components and states. Finally, the relationship between components and states is quantified by defining and calculating the linkage of components to states. The relationship between components and states is found to be both intuitive and paradoxical, depending on the ratios of the state lifetimes. Nevertheless, both the intuitive and paradoxical observations can be described within a consistent framework. The approach used here allows the exponential components to be interpreted in terms of underlying states for all possible values of the rate constants, something not previously possible.

A Comment on Ion Channels as Pharmacological Targets in Oncology

Becchetti, A., Arcangeli, A. — 2008-07-28