The Journal of General Physiology recent issues

The outer segment serves as a default destination for the trafficking of membrane proteins in photoreceptors

2008-11-24

Selective coupling of type 6 adenylyl cyclase with type 2 IP3 receptors mediates direct sensitization of IP3 receptors by cAMP

Tovey, S. C., Dedos, S. G., Taylor, E. J.A., Church, J. E., Taylor, C. W. — 2008-11-24

The Two-Membrane Model of Epithelial Transport: Koefoed-Johnsen and Ussing (1958)

Palmer, L. G., Andersen, O. S. — 2008-11-24

Long-pore Electrostatics in Inward-rectifier Potassium Channels

Robertson, J. L., Palmer, L. G., Roux, B. — 2008-11-24

Inward-rectifier potassium (Kir) channels differ from the canonical K⁺ channel structure in that they possess a long extended pore (~85 Å) for ion conduction that reaches deeply into the cytoplasm. This unique structural feature is presumably involved in regulating functional properties specific to Kir channels, such as conductance, rectification block, and ligand-dependent gating. To elucidate the underpinnings of these functional roles, we examine the electrostatics of an ion along this extended pore. Homology models are constructed based on the open-state model of KirBac1.1 for four mammalian Kir channels: Kir1.1/ROMK, Kir2.1/IRK, Kir3.1/GIRK, and Kir6.2/KATP. By solving the Poisson-Boltzmann equation, the electrostatic free energy of a K⁺ ion is determined along each pore, revealing that mammalian Kir channels provide a favorable environment for cations and suggesting the existence of high-density regions in the cytoplasmic domain and cavity. The contribution from the reaction field (the self-energy arising from the dielectric polarization induced by the ion's charge in the complex geometry of the pore) is unfavorable inside the long pore. However, this is well compensated by the electrostatic interaction with the static field arising from the protein charges and shielded by the dielectric surrounding. Decomposition of the static field provides a list of residues that display remarkable correspondence with existing mutagenesis data identifying amino acids that affect conduction and rectification. Many of these residues demonstrate interactions with the ion over long distances, up to 40 Å, suggesting that mutations potentially affect ion or blocker energetics over the entire pore. These results provide a foundation for understanding ion interactions in Kir channels and extend to the study of ion permeation, block, and gating in long, cation-specific pores.

Slow Inactivation in Shaker K Channels Is Delayed by Intracellular Tetraethylammonium

2008-11-24

After removal of the fast N-type inactivation gate, voltage-sensitive Shaker (Shaker IR) K channels are still able to inactivate, albeit slowly, upon sustained depolarization. The classical mechanism proposed for the slow inactivation observed in cell-free membrane patches—the so called C inactivation—is a constriction of the external mouth of the channel pore that prevents K⁺ ion conduction. This constriction is antagonized by the external application of the pore blocker tetraethylammonium (TEA). In contrast to C inactivation, here we show that, when recorded in whole Xenopus oocytes, slow inactivation kinetics in Shaker IR K channels is poorly dependent on external TEA but severely delayed by internal TEA. Based on the antagonism with internally or externally added TEA, we used a two-pulse protocol to show that half of the channels inactivate by way of a gate sensitive to internal TEA. Such gate had a recovery time course in the tens of milliseconds range when the interpulse voltage was –90 mV, whereas C-inactivated channels took several seconds to recover. Internal TEA also reduced gating charge conversion associated to slow inactivation, suggesting that the closing of the internal TEA-sensitive inactivation gate could be associated with a significant amount of charge exchange of this type. We interpreted our data assuming that binding of internal TEA antagonized with U-type inactivation (Klemic, K.G., G.E. Kirsch, and S.W. Jones. 2001. Biophys. J. 81:814–826). Our results are consistent with a direct steric interference of internal TEA with an internally located slow inactivation gate as a "foot in the door" mechanism, implying a significant functional overlap between the gate of the internal TEA-sensitive slow inactivation and the primary activation gate. But, because U-type inactivation is reduced by channel opening, trapping the channel in the open conformation by TEA would also yield to an allosteric delay of slow inactivation. These results provide a framework to explain why constitutively C-inactivated channels exhibit gating charge conversion, and why mutations at the internal exit of the pore, such as those associated to episodic ataxia type I in hKv1.1, cause severe changes in inactivation kinetics.

Molecular Template for a Voltage Sensor in a Novel K+ Channel. III. Functional Reconstitution of a Sensorless Pore Module from a Prokaryotic Kv Channel

Santos, J. S., Grigoriev, S. M., Montal, M. — 2008-11-24

KvLm is a prokaryotic voltage-gated K⁺ (Kv) channel from Listeria monocytogenes. The sequence of the voltage-sensing module (transmembrane segments S1-S4) of KvLm is atypical in that it contains only three of the eight conserved charged residues known to be deterministic for voltage sensing in eukaryotic Kv's. In contrast, the pore module (PM), including the S4-S5 linker and cytoplasmic tail (linker-S5-P-S6-C-terminus) of KvLm, is highly conserved. Here, the full-length (FL)-KvLm and the KvLm-PM only proteins were expressed, purified, and reconstituted into giant liposomes. The properties of the reconstituted FL-KvLm mirror well the characteristics of the heterologously expressed channel in Escherichia coli spheroplasts: a right-shifted voltage of activation, micromolar tetrabutylammonium-blocking affinity, and a single-channel conductance comparable to that of eukaryotic Kv's. Conversely, ionic currents through the PM recapitulate both the conductance and blocking properties of the FL-KvLm, yet the KvLm-PM exhibits only rudimentary voltage dependence. Given that the KvLm-PM displays many of the conduction properties of FL-KvLm and of other eukaryotic Kv's, including strict ion selectivity, we conclude that self-assembly of the PM subunits in lipid bilayers, in the absence of the voltage-sensing module, generates a conductive oligomer akin to that of the native KvLm, and that the structural independence of voltage sensing and PMs observed in eukaryotic Kv channels was initially implemented by nature in the design of prokaryotic Kv channels. Collectively, the results indicate that this robust functional module will prove valuable as a molecular template for coupling new sensors and to elucidate PM residue–specific contributions to Kv conduction properties.

Kv Channel Gating Requires a Compatible S4-S5 Linker and Bottom Part of S6, Constrained by Non-interacting Residues

2008-11-24

Voltage-dependent K⁺ channels transfer the voltage sensor movement into gate opening or closure through an electromechanical coupling. To test functionally whether an interaction between the S4-S5 linker (L45) and the cytoplasmic end of S6 (S6_T) constitutes this coupling, the L45 in hKv1.5 was replaced by corresponding hKv2.1 sequence. This exchange was not tolerated but could be rescued by also swapping S6_T. Exchanging both L45 and S6_T transferred hKv2.1 kinetics to an hKv1.5 background while preserving the voltage dependence. A one-by-one residue substitution scan of L45 and S6_T in hKv1.5 further shows that S6_T needs to be -helical and forms a "crevice" in which residues I422 and T426 of L45 reside. These residues transfer the mechanical energy onto the S6_T crevice, whereas other residues in S6_T and L45 that are not involved in the interaction maintain the correct structure of the coupling.

Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

Xiao, Q., Prussia, A., Yu, K., Cui, Y.-y., Hartzell, H. C. — 2008-11-24

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl^– ion channels that are activated by intracellular Ca²⁺. At present, the structures and mechanisms responsible for Ca²⁺ sensing remain unknown. Here, we have used a combination of molecular modeling, density functional–binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca²⁺ sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca²⁺ sensing by hBest1. The cluster of five amino acids (293–308) is crucial for normal channel gating by Ca²⁺ because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca²⁺. Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca²⁺ with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca²⁺-binding loop (312–323) reduced the apparent Ca²⁺ affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca²⁺-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350–390) that is required for Ca²⁺ activation and plays a role in current rundown. These experiments identify a region of hBest1 (312–323) that is involved in the gating of hBest1 by Ca²⁺ and suggest a model in which Ca²⁺ binding to EF1 activates the channel in a process that requires the acidic domain (293–308) and another regulatory domain (350–390). Many of the ~100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca²⁺ sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca²⁺.

Identification of Channel-lining Amino Acid Residues in the Hydrophobic Segment of Colicin Ia

Kienker, P. K., Jakes, K. S., Finkelstein, A. — 2008-11-24

Colicin Ia is a bactericidal protein of 626 amino acid residues that kills its target cell by forming a channel in the inner membrane; it can also form voltage-dependent channels in planar lipid bilayer membranes. The channel-forming activity resides in the carboxy-terminal domain of ~177 residues. In the crystal structure of the water-soluble conformation, this domain consists of a bundle of 10 -helices, with eight mostly amphipathic helices surrounding a hydrophobic helical hairpin (helices H8-H9). We wish to know how this structure changes to form a channel in a lipid bilayer. Although there is evidence that the open channel has four transmembrane segments (H8, H9, and parts of H1 and H6-H7), their arrangement relative to the pore is largely unknown. Given the lack of a detailed structural model, it is imperative to better characterize the channel-lining protein segments. Here, we focus on a segment of 44 residues (573–616), which in the crystal structure comprises the H8-H9 hairpin and flanking regions. We mutated each of these residues to a unique cysteine, added the mutant colicins to the cis side of planar bilayers to form channels, and determined whether sulfhydryl-specific methanethiosulfonate reagents could alter the conduction of ions through the open channel. We found a pattern of reactivity consistent with parts of H8 and H9 lining the channel as -helices, albeit rather short ones for spanning a lipid bilayer (12 residues). The effects of the reactions on channel conductance and selectivity tend to be greater for residues near the amino terminus of H8 and the carboxy terminus of H9, with particularly large effects for G577C, T581C, and G609C, suggesting that these residues may occupy a relatively constricted region near the cis end of the channel.

The Roles of Pore Ring and Plug in the SecY Protein-conducting Channel

Gumbart, J., Schulten, K. — 2008-11-24

The protein-conducting channel, or translocon, is an evolutionarily conserved complex that allows nascent proteins to cross a cellular membrane or integrate into it. The crystal structure of an archaeal translocon, the SecY complex, revealed that two elements contribute to sealing the channel: a small "plug" domain blocking the periplasmic region of the channel, and a pore ring composed of six hydrophobic residues acting as a constriction point at the channel's center. To determine the independent functions of these two elements, we have performed molecular dynamics simulations of the native channel as well as of two recently structurally resolved mutants in which portions of their plugs were deleted. We find that in the mutants, the instability in the plug region leads to a concomitant increase in flexibility of the pore ring. The instability is quantified by the rate of water permeation in each system as well as by the force required for oligopeptide translocation. Through a novel simulation in which the interactions between the plug and water were independently controlled, we find that the role of the plug in stabilizing the pore ring is significantly more important than its role as a purely steric barrier.

Modulation of the Local SR Ca2+ Release by Intracellular Mg2+ in Cardiac Myocytes

Gusev, K., Niggli, E. — 2008-11-24

In cardiac muscle, Ca²⁺-induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) defines the amplitude and time course of the Ca²⁺ transient. The global elevation of the intracellular Ca²⁺ concentration arises from the spatial and temporal summation of elementary Ca²⁺ release events, Ca²⁺ sparks. Ca²⁺ sparks represent the concerted opening of a group of ryanodine receptors (RYRs), which are under the control of several modulatory proteins and diffusible cytoplasmic factors (e.g., Ca²⁺, Mg²⁺, and ATP). Here, we examined by which mechanism the free intracellular Mg²⁺ ([Mg²⁺]_free) affects various Ca²⁺ spark parameters in permeabilized mouse ventricular myocytes, such as spark frequency, duration, rise time, and full width, at half magnitude and half maximal duration. Varying the levels of free ATP and Mg²⁺ in specifically designed solutions allowed us to separate the inhibition of RYRs by Mg²⁺ from the possible activation by ATP and Mg²⁺-ATP via the adenine binding site of the channel. Changes in [Mg²⁺]_free generally led to biphasic alterations of the Ca²⁺ spark frequency. For example, lowering [Mg²⁺]_free resulted in an abrupt increase of spark frequency, which slowly recovered toward the initial level, presumably as a result of SR Ca²⁺ depletion. Fitting the Ca²⁺ spark inhibition by [Mg²⁺]_free with a Hill equation revealed a K_i of 0.1 mM. In conclusion, our results support the notion that local Ca²⁺ release and Ca²⁺ sparks are modulated by Mg²⁺ in the intracellular environment. This seems to occur predominantly by hindering Ca²⁺-dependent activation of the RYRs through competitive Mg²⁺ occupancy of the high-affinity activation site of the channels. These findings help to characterize CICR in cardiac muscle under normal and pathological conditions, where the levels of Mg²⁺ and ATP can change.

Voltage Dependence of ATP Secretion in Mammalian Taste Cells

Romanov, R. A., Rogachevskaja, O. A., Khokhlov, A. A., Kolesnikov, S. S. — 2008-11-24

Mammalian type II taste cells release the afferent neurotransmitter adenosine triphosphate (ATP) through ATP-permeable ion channels, most likely to be connexin (Cx) and/or pannexin hemichannels. Here, we show that ion channels responsible for voltage-gated (VG) outward currents in type II cells are ATP permeable and demonstrate a strong correlation between the magnitude of the VG current and the intensity of ATP release. These findings suggest that slowly deactivating ion channels transporting the VG outward currents can also mediate ATP secretion in type II cells. In line with this inference, we studied a dependence of ATP secretion on membrane voltage with a cellular ATP sensor using different pulse protocols. These were designed on the basis of predictions of a model of voltage-dependent transient ATP efflux. Consistently with curves that were simulated for ATP release mediated by ATP-permeable channels deactivating slowly, the bell-like and Langmuir isotherm–like potential dependencies were characteristic of ATP secretion obtained for prolonged and short electrical stimulations of taste cells, respectively. These observations strongly support the idea that ATP is primarily released via slowly deactivating channels. Depolarizing voltage pulses produced negligible Ca²⁺ transients in the cytoplasm of cells releasing ATP, suggesting that ATP secretion is mainly governed by membrane voltage under our recording conditions. With the proviso that natural connexons and pannexons are kinetically similar to exogenously expressed hemichannels, our findings suggest that VG ATP release in type II cells is primarily mediated by Cx hemichannels.

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.