The Journal of General Physiology Articles

Mechanosensitive Channel MscS in the Open State: Modeling of the Transition, Explicit Simulations, and Experimental Measurements of Conductance

Anishkin, A., Kamaraju, K., Sukharev, S. — 2008-06-30

Mechanosensitive channels of small conductance (MscS) are ubiquitous turgor pressure regulators found in many walled cells and some intracellular organelles. Escherichia coli MscS acting as a tension-activated osmolyte release valve shows a nonsaturable conductance (1.2 nS in a 39 mS/cm electrolyte) and weak preference for anions. Pursuing the transition pathways in this channel, we applied the extrapolated motion protocol (cycles of displacements, minimizations, and short simulations) to the previously generated compact resting conformation of MscS. We observed tilting and straightening of the kinked pore-forming TM3 helices during the barrel expansion. Extended all-atom simulations confirmed the stability of the open conformation in the bilayer. A 53° spontaneous axial rotation of TM3s observed after equilibration increased the width and polarity of the pore allowing for stable voltage-independent hydration and presence of both cations and anions throughout the pore. The resultant open state, characterized by a pore 1.6 nm wide, satisfied the experimental conductance and in-plane expansion. Applied transmembrane electric field (±100 to ±200 mV) in simulations produced a flow of both K⁺ and Cl^–, with Cl^– current dominating at higher voltages. Electroosmotic water flux strongly correlated with the chloride current (~8 waters per Cl^–). The selectivity and rectification were in agreement with the experimental measurements performed in the same range of voltages. Among the charged residues surrounding the pore, only K169 was found to contribute noticeably in the rectification. We conclude that (a) the barrel expansion involving tilting, straightening, and rotation of TM3s provides the geometry and electrostatics that accounts for the conductive properties of the open pore; (b) the observed regimen of ion passage through the pore is similar to electrodiffusion, thus macroscopic estimations closely approximate the experimental and molecular dynamics-simulated conductances; (c) increased interaction of the opposing ionic fluxes at higher voltages may result in selectivities stronger than measured near the reversal potential.

How ATP Inhibits the Open KATP Channel

Craig, T. J., Ashcroft, F. M., Proks, P. — 2008-06-30

ATP-sensitive potassium (K_ATP) channels are composed of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits. Binding of ATP to Kir6.2 leads to inhibition of channel activity. Because there are four subunits and thus four ATP-binding sites, four binding events are possible. ATP binds to both the open and closed states of the channel and produces a decrease in the mean open time, a reduction in the mean burst duration, and an increase in the frequency and duration of the interburst closed states. Here, we investigate the mechanism of interaction of ATP with the open state of the channel by analyzing the single-channel kinetics of concatenated Kir6.2 tetramers containing from zero to four mutated Kir6.2 subunits that possess an impaired ATP-binding site. We show that the ATP-dependent decrease in the mean burst duration is well described by a Monod-Wyman-Changeux model in which channel closing is produced by all four subunits acting in a single concerted step. The data are inconsistent with a Hodgkin-Huxley model (four independent steps) or a dimer model (two independent dimers). When the channel is open, ATP binds to a single ATP-binding site with a dissociation constant of 300 µM.

HCO3- Secretion by Murine Nasal Submucosal Gland Serous Acinar Cells during Ca2+-stimulated Fluid Secretion

Lee, R. J., Harlow, J. M., Limberis, M. P., Wilson, J. M., Foskett, J. K. — 2008-06-30

Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca²⁺-activated Cl^– secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca²⁺-activated Cl^– secretion was accompanied by secretion of HCO₃^–, possibly a critical ASL component, by simultaneous measurements of intracellular pH (pH_i) and cell volume. Resting pH_i was 7.17 ± 0.01 in physiological medium (5% CO₂–25 mM HCO₃^–). During carbachol (CCh) stimulation, pH_i fell transiently by 0.08 ± 0.01 U concomitantly with a fall in Cl^– content revealed by cell shrinkage, reflecting Cl^– secretion. A subsequent alkalinization elevated pH_i to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO₂–HCO₃^–-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO₃^– efflux by ion substitution or exposure to the Cl^– channel inhibitor niflumic acid (100 µM) strongly inhibited agonist-induced acidification by >80% and >70%, respectively. The Na⁺/H⁺ exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidification. Gene expression profiling suggested that serous cells express NHE isoforms 1–4 and 6–9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pH_i recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO₃^– during Ca²⁺-evoked fluid secretion by a mechanism that involves the apical membrane secretory Cl^– channel, with HCO₃^– secretion sustained by activation of NHE1 in the basolateral membrane. In addition, other Na⁺-dependent pH_i regulatory mechanisms exist, as evidenced by stronger inhibition of alkalinization in Na⁺-free media.

Direct Regulation of BK Channels by Phosphatidylinositol 4,5-Bisphosphate as a Novel Signaling Pathway

2008-06-30

Large conductance, calcium- and voltage-gated potassium (BK) channels are ubiquitous and critical for neuronal function, immunity, and smooth muscle contractility. BK channels are thought to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP₂) only through phospholipase C (PLC)–generated PIP₂ metabolites that target Ca²⁺ stores and protein kinase C and, eventually, the BK channel. Here, we report that PIP₂ activates BK channels independently of PIP₂ metabolites. PIP₂ enhances Ca²⁺-driven gating and alters both open and closed channel distributions without affecting voltage gating and unitary conductance. Recovery from activation was strongly dependent on PIP₂ acyl chain length, with channels exposed to water-soluble diC4 and diC8 showing much faster recovery than those exposed to PIP₂ (diC16). The PIP₂–channel interaction requires negative charge and the inositol moiety in the phospholipid headgroup, and the sequence RKK in the S6–S7 cytosolic linker of the BK channel-forming (cbv1) subunit. PIP₂-induced activation is drastically potentiated by accessory β₁ (but not β₄) channel subunits. Moreover, PIP₂ robustly activates BK channels in vascular myocytes, where β₁ subunits are abundantly expressed, but not in skeletal myocytes, where these subunits are barely detectable. These data demonstrate that the final PIP₂ effect is determined by channel accessory subunits, and such mechanism is subunit specific. In HEK293 cells, cotransfection of cbv1+β₁ and PI4-kinaseII robustly activates BK channels, suggesting a role for endogenous PIP₂ in modulating channel activity. Indeed, in membrane patches excised from vascular myocytes, BK channel activity runs down and Mg-ATP recovers it, this recovery being abolished by PIP₂ antibodies applied to the cytosolic membrane surface. Moreover, in intact arterial myocytes under physiological conditions, PLC inhibition on top of blockade of downstream signaling leads to drastic BK channel activation. Finally, pharmacological treatment that raises PIP₂ levels and activates BK channels dilates de-endothelized arteries that regulate cerebral blood flow. These data indicate that endogenous PIP₂ directly activates vascular myocyte BK channels to control vascular tone.

Massive Ca-induced Membrane Fusion and Phospholipid Changes Triggered by Reverse Na/Ca Exchange in BHK Fibroblasts

2008-06-30

Baby hamster kidney (BHK) fibroblasts increase their cell capacitance by 25–100% within 5 s upon activating maximal Ca influx via constitutively expressed cardiac Na/Ca exchangers (NCX1). Free Ca, measured with fluo-5N, transiently exceeds 0.2 mM with total Ca influx amounting to ~5 mmol/liter cell volume. Capacitance responses are half-maximal when NCX1 promotes a free cytoplasmic Ca of 0.12 mM (Hill coefficient 2). Capacitance can return to baseline in 1–3 min, and responses can be repeated several times. The membrane tracer, FM 4-64, is taken up during recovery and can be released at a subsequent Ca influx episode. Given recent interest in signaling lipids in membrane fusion, we used green fluorescent protein (GFP) fusions with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and diacylglycerol (DAG) binding domains to analyze phospholipid changes in relation to these responses. PI(4,5)P₂ is rapidly cleaved upon activating Ca influx and recovers within 2 min. However, PI(4,5)P₂ depletion by activation of overexpressed hM1 muscarinic receptors causes only little membrane fusion, and subsequent fusion in response to Ca influx remains massive. Two results suggest that DAG may be generated from sources other than PI(4,5)P in these protocols. First, acylglycerols are generated in response to elevated Ca, even when PI(4,5)P₂ is metabolically depleted. Second, DAG-binding C1A-GFP domains, which are brought to the cell surface by exogenous ligands, translocate rapidly back to the cytoplasm in response to Ca influx. Nevertheless, inhibitors of PLCs and cPLA2, PI(4,5)P₂-binding peptides, and PLD modification by butanol do not block membrane fusion. The cationic agents, FM 4-64 and heptalysine, bind profusely to the extracellular cell surface during membrane fusion. While this binding might reflect phosphatidylserine (PS) "scrambling" between monolayers, it is unaffected by a PS-binding protein, lactadherin, and by polylysine from the cytoplasmic side. Furthermore, the PS indicator, annexin-V, binds only slowly after fusion. Therefore, we suggest that the luminal surfaces of membrane vesicles that fuse to the plasmalemma may be rather anionic. In summary, our results provide no support for any regulatory or modulatory role of phospholipids in Ca-induced membrane fusion in fibroblasts.

Ca-dependent Nonsecretory Vesicle Fusion in a Secretory Cell

Wang, T.-M., Hilgemann, D. W. — 2008-06-30

We have compared Ca-dependent exocytosis in excised giant membrane patches and in whole-cell patch clamp with emphasis on the rat secretory cell line, RBL. Stable patches of 2–4 pF are easily excised from RBL cells after partially disrupting actin cytoskeleton with latrunculin A. Membrane fusion is triggered by switching the patch to a cytoplasmic solution containing 100–200 µM free Ca. Capacitance and amperometric recording show that large secretory granules (SGs) containing serotonin are mostly lost from patches. Small vesicles that are retained (non-SGs) do not release serotonin or other substances detected by amperometry, although their fusion is reduced by tetanus toxin light chain. Non-SG fusion is unaffected by N-ethylmaleimide, phosphatidylinositol-4,5-bis-phosphate (PI(4,5)P₂) ligands, such as neomycin, a PI-transfer protein that can remove PI from membranes, the PI(3)-kinase inhibitor LY294002 and PI(4,5)P₂, PI(3)P, and PI(4)P antibodies. In patch recordings, but not whole-cell recordings, fusion can be strongly reduced by ATP removal and by the nonspecific PI-kinase inhibitors wortmannin and adenosine. In whole-cell recording, non-SG fusion is strongly reduced by osmotically induced cell swelling, and subsequent recovery after shrinkage is then inhibited by wortmannin. Thus, membrane stretch that occurs during patch formation may be a major cause of differences between excised patch and whole-cell fusion responses. Regarding Ca sensors for non-SG fusion, fusion remains robust in synaptotagmin (Syt) VII–/– mouse embryonic fibroblasts (MEFs), as well as in PLC1, PLC 1/4, and PLC1–/– MEFs. Thus, Syt VII and several PLCs are not required. Furthermore, the Ca dependence of non-SG fusion reflects a lower Ca affinity (K_D ~71 µM) than expected for these C2 domain–containing proteins. In summary, we find that non-SG membrane fusion behaves and is regulated substantially differently from SG fusion, and we have identified an ATP-dependent process that restores non-SG fusion capability after it is perturbed by membrane stretch or cell dilation.

Amino Acid Substitutions in the Pore Helix of GluR6 Control Inhibition by Membrane Fatty Acids

Wilding, T. J., Fulling, E., Zhou, Y., Huettner, J. E. — 2008-06-30

RNA editing at the Q/R site in the GluR5 and GluR6 subunits of neuronal kainate receptors regulates channel inhibition by lipid-derived modulators including the cis-unsaturated fatty acids arachidonic acid and docosahexaenoic acid. Kainate receptor channels in which all of the subunits are in the edited (R) form exhibit strong inhibition by these compounds, whereas wild-type receptors that include a glutamine (Q) at the Q/R site in one or more subunits are resistant to inhibition. In the present study, we have performed an arginine scan of residues in the pore loop of the GluR6(Q) subunit. Amino acids within the range from –19 to +7 of the Q/R site of GluR6(Q) were individually mutated to arginine and the mutant cDNAs were expressed as homomeric channels in HEK 293 cells. All but one of the single arginine substitution mutants yielded functional channels. Only weak inhibition, typical of wild-type GluR6(Q) channels, was observed for substitutions +1 to +6 downstream of the Q/R site. However, arginine substitution at several locations upstream of the Q/R site resulted in homomeric channels exhibiting strong inhibition by fatty acids, which is characteristic of homomeric GluR6(R) channels. Based on homology with the pore loop of potassium channels, locations at which R substitution induces susceptibility to fatty acid inhibition face away from the cytoplasm toward the M1 and M3 helices and surrounding lipids.

Differential Interactions of Na+ Channel Toxins with T-type Ca2+ Channels

2008-06-30

Two types of voltage-dependent Ca²⁺ channels have been identified in heart: high (I_CaL) and low (I_CaT) voltage-activated Ca²⁺ channels. In guinea pig ventricular myocytes, low voltage–activated inward current consists of I_CaT and a tetrodotoxin (TTX)-sensitive I_Ca component (I_Ca(TTX)). In this study, we reexamined the nature of low-threshold I_Ca in dog atrium, as well as whether it is affected by Na⁺ channel toxins. Ca²⁺ currents were recorded using the whole-cell patch clamp technique. In the absence of external Na⁺, a transient inward current activated near –50 mV, peaked at –30 mV, and reversed around +40 mV (HP = –90 mV). It was unaffected by 30 µM TTX or micromolar concentrations of external Na⁺, but was inhibited by 50 µM Ni²⁺ (by ~90%) or 5 µM mibefradil (by ~50%), consistent with the reported properties of I_CaT. Addition of 30 µM TTX in the presence of Ni²⁺ increased the current approximately fourfold (41% of control), and shifted the dose–response curve of Ni²⁺ block to the right (IC₅₀ from 7.6 to 30 µM). Saxitoxin (STX) at 1 µM abolished the current left in 50 µM Ni²⁺. In the absence of Ni²⁺, STX potently blocked I_CaT (EC₅₀ = 185 nM) and modestly reduced I_CaL (EC₅₀ = 1.6 µM). While TTX produced no direct effect on I_CaT elicited by expression of hCa_V3.1 and hCa_V3.2 in HEK-293 cells, it significantly attenuated the block of this current by Ni²⁺ (IC₅₀ increased to 550 µM Ni²⁺ for Ca_V3.1 and 15 µM Ni²⁺ for Ca_V3.2); in contrast, 30 µM TTX directly inhibited hCa_V3.3-induced I_CaT and the addition of 750 µM Ni²⁺ to the TTX-containing medium led to greater block of the current that was not significantly different than that produced by Ni²⁺ alone. 1 µM STX directly inhibited Ca_V3.1-, Ca_V3.2-, and Ca_V3.3-mediated I_CaT but did not enhance the ability of Ni²⁺ to block these currents. These findings provide important new implications for our understanding of structure–function relationships of I_CaT in heart, and further extend the hypothesis of a parallel evolution of Na⁺ and Ca²⁺ channels from an ancestor with common structural motifs.

Species-specific Differences among KCNMB3 BK {beta}3 Auxiliary Subunits: Some {beta}3 N-terminal Variants May Be Primate-specific Subunits

Zeng, X., Xia, X.-M., Lingle, C. J. — 2008-06-30

The KCNMB3 gene encodes one of a family of four auxiliary β subunits found in the mammalian genome that associate with Slo1 subunits and regulate BK channel function. In humans, the KCNMB3 gene contains four N-terminal alternative exons that produce four functionally distinct β3 subunits, β3a–d. Three variants, β3a–c, exhibit kinetically distinct inactivation behaviors. Since investigation of the physiological roles of BK auxiliary subunits will depend on studies in rodents, here we have determined the identity and functional properties of mouse β3 variants. Whereas β1, β2, and β4 subunits exhibit 83.2%, 95.3%, and 93.8% identity between mouse and human, the mouse β3 subunit, excluding N-terminal splice variants, shares only 62.8% amino acid identity with its human counterpart. Based on an examination of the mouse genome and screening of mouse cDNA libraries, here we have identified only two N-terminal candidates, β3a and β3b, of the four found in humans. Both human and mouse β3a subunits produce a characteristic use-dependent inactivation. Surprisingly, whereas the hβ3b exhibits rapid inactivation, the putative mβ3b does not inactivate. Furthermore, unlike hβ3, the mβ3 subunit, irrespective of the N terminus, mediates a shift in gating to more negative potentials at a given Ca²⁺ concentration. The shift in gating gradually is lost following patch excision, suggesting that the gating shift involves some regulatory process dependent on the cytosolic milieu. Examination of additional genomes to assess conservation among splice variants suggests that the putative mβ3b N terminus may not be a true orthologue of the hβ3b N terminus and that both β3c and β3d appear likely to be primate-specific N-terminal variants. These results have three key implications: first, functional properties of homologous β3 subunits may differ among mammalian species; second, the specific physiological roles of homologous β3 subunits may differ among mammalian species; and, third, some β3 variants may be primate-specific ion channel subunits.

A Close Association of RyRs with Highly Dense Clusters of Ca2+-activated Cl- Channels Underlies the Activation of STICs by Ca2+ Sparks in Mouse Airway Smooth Muscle

Bao, R., Lifshitz, L. M., Tuft, R. A., Bellve, K., Fogarty, K. E., ZhuGe, R. — 2008-06-30

Ca²⁺ sparks are highly localized, transient releases of Ca²⁺ from sarcoplasmic reticulum through ryanodine receptors (RyRs). In smooth muscle, Ca²⁺ sparks trigger spontaneous transient outward currents (STOCs) by opening nearby clusters of large-conductance Ca²⁺-activated K⁺ channels, and also gate Ca²⁺-activated Cl^– (Cl_(Ca)) channels to induce spontaneous transient inward currents (STICs). While the molecular mechanisms underlying the activation of STOCs by Ca²⁺ sparks is well understood, little information is available on how Ca²⁺ sparks activate STICs. In the present study, we investigated the spatial organization of RyRs and Cl_(Ca) channels in spark sites in airway myocytes from mouse. Ca²⁺ sparks and STICs were simultaneously recorded, respectively, with high-speed, widefield digital microscopy and whole-cell patch-clamp. An image-based approach was applied to measure the Ca²⁺ current underlying a Ca²⁺ spark (I_Ca(spark)), with an appropriate correction for endogenous fixed Ca²⁺ buffer, which was characterized by flash photolysis of NPEGTA. We found that I_Ca(spark) rises to a peak in 9 ms and decays with a single exponential with a time constant of 12 ms, suggesting that Ca²⁺ sparks result from the nonsimultaneous opening and closure of multiple RyRs. The onset of the STIC lags the onset of the I_Ca(spark) by less than 3 ms, and its rising phase matches the duration of the I_Ca(spark). We further determined that Cl_(Ca) channels on average are exposed to a [Ca²⁺] of 2.4 µM or greater during Ca²⁺ sparks. The area of the plasma membrane reaching this level is <600 nm in radius, as revealed by the spatiotemporal profile of [Ca²⁺] produced by a reaction-diffusion simulation with measured I_Ca(spark). Finally we estimated that the number of Cl_(Ca) channels localized in Ca²⁺ spark sites could account for all the Cl_(Ca) channels in the entire cell. Taken together these results lead us to propose a model in which RyRs and Cl_(Ca) channels in Ca²⁺ spark sites localize near to each other, and, moreover, Cl_(Ca) channels concentrate in an area with a radius of ~600 nm, where their density reaches as high as 300 channels/µm². This model reveals that Cl_(Ca) channels are tightly controlled by Ca²⁺ sparks via local Ca²⁺ signaling.

Intracellular Proton Regulation of ClC-0

Zifarelli, G., Murgia, A. R., Soliani, P., Pusch, M. — 2008-06-30

Some CLC proteins function as passive Cl^– ion channels whereas others are secondary active chloride/proton antiporters. Voltage-dependent gating of the model Torpedo channel ClC-0 is modulated by intracellular and extracellular pH, possibly reflecting a mechanistic relationship with the chloride/proton coupling of CLC antiporters. We used inside-out patch clamp measurements and mutagenesis to explore the dependence of the fast gating mechanism of ClC-0 on intracellular pH and to identify the putative intracellular proton acceptor(s). Among the tested residues (S123, K129, R133, K149, E166, F214L, S224, E226, V227, C229, R305, R312, C415, H472, F418, V419, P420, and Y512) only mutants of E166, F214, and F418 qualitatively changed the pH_int dependence. No tested amino acid emerged as a valid candidate for being a pH sensor. A detailed kinetic analysis of the dependence of fast gate relaxations on pH_int and [Cl^–]_int provided quantitative constraints on possible mechanistic models of gating. In one particular model, a proton is generated by the dissociation of a water molecule in an intrapore chloride ion binding site. The proton is delivered to the side chain of E166 leading to the opening of the channel, while the hydroxyl ion is stabilized in the internal/central anion binding site. Deuterium isotope effects confirm that proton transfer is rate limiting for fast gate opening and that channel closure depends mostly on the concentration of OH^– ions. The gating model is in natural agreement with the finding that only the closing rate constant, but not the opening rate constant, depends on pH_int and [Cl^–]_int.

Position and Role of the BK Channel {alpha} Subunit S0 Helix Inferred from Disulfide Crosslinking

2008-05-26

The position and role of the unique N-terminal transmembrane (TM) helix, S0, in large-conductance, voltage- and calcium-activated potassium (BK) channels are undetermined. From the extents of intra-subunit, endogenous disulfide bond formation between cysteines substituted for the residues just outside the membrane domain, we infer that the extracellular flank of S0 is surrounded on three sides by the extracellular flanks of TM helices S1 and S2 and the four-residue extracellular loop between S3 and S4. Eight different double cysteine–substituted alphas, each with one cysteine in the S0 flank and one in the S3–S4 loop, were at least 90% disulfide cross-linked. Two of these alphas formed channels in which 90% cross-linking had no effect on the V₅₀ or on the activation and deactivation rate constants. This implies that the extracellular ends of S0, S3, and S4 are close in the resting state and move in concert during voltage sensor activation. The association of S0 with the gating charge bearing S3 and S4 could contribute to the considerably larger electrostatic energy required to activate the BK channel compared with typical voltage-gated potassium channels with six TM helices.

Atomic Constraints between the Voltage Sensor and the Pore Domain in a Voltage-gated K+ Channel of Known Structure

Lewis, A., Jogini, V., Blachowicz, L., Laine, M., Roux, B. — 2008-05-26

In voltage-gated K⁺ channels (Kv), membrane depolarization promotes a structural reorganization of each of the four voltage sensor domains surrounding the conducting pore, inducing its opening. Although the crystal structure of Kv1.2 provided the first atomic resolution view of a eukaryotic Kv channel, several components of the voltage sensors remain poorly resolved. In particular, the position and orientation of the charged arginine side chains in the S4 transmembrane segments remain controversial. Here we investigate the proximity of S4 and the pore domain in functional Kv1.2 channels in a native membrane environment using electrophysiological analysis of intersubunit histidine metallic bridges formed between the first arginine of S4 (R294) and residues A351 or D352 of the pore domain. We show that histidine pairs are able to bind Zn²⁺ or Cd²⁺ with high affinity, demonstrating their close physical proximity. The results of molecular dynamics simulations, consistent with electrophysiological data, indicate that the position of the S4 helix in the functional open-activated state could be shifted by ~7–8 Å and rotated counterclockwise by 37° along its main axis relative to its position observed in the Kv1.2 x-ray structure. A structural model is provided for this conformation. The results further highlight the dynamic and flexible nature of the voltage sensor.

A Continuum Method for Determining Membrane Protein Insertion Energies and the Problem of Charged Residues

Choe, S., Hecht, K. A., Grabe, M. — 2008-05-26

Continuum electrostatic approaches have been extremely successful at describing the charged nature of soluble proteins and how they interact with binding partners. However, it is unclear whether continuum methods can be used to quantitatively understand the energetics of membrane protein insertion and stability. Recent translation experiments suggest that the energy required to insert charged peptides into membranes is much smaller than predicted by present continuum theories. Atomistic simulations have pointed to bilayer inhomogeneity and membrane deformation around buried charged groups as two critical features that are neglected in simpler models. Here, we develop a fully continuum method that circumvents both of these shortcomings by using elasticity theory to determine the shape of the deformed membrane and then subsequently uses this shape to carry out continuum electrostatics calculations. Our method does an excellent job of quantitatively matching results from detailed molecular dynamics simulations at a tiny fraction of the computational cost. We expect that this method will be ideal for studying large membrane protein complexes.

NGF Inhibits M/KCNQ Currents and Selectively Alters Neuronal Excitability in Subsets of Sympathetic Neurons Depending on their M/KCNQ Current Background

Jia, Z., Bei, J., Rodat-Despoix, L., Liu, B., Jia, Q., Delmas, P., Zhang, H. — 2008-05-26

M/KCNQ currents play a critical role in the determination of neuronal excitability. Many neurotransmitters and peptides modulate M/KCNQ current and neuronal excitability through their G protein–coupled receptors. Nerve growth factor (NGF) activates its receptor, a member of receptor tyrosine kinase (RTK) superfamily, and crucially modulates neuronal cell survival, proliferation, and differentiation. In this study, we studied the effect of NGF on the neuronal (rat superior cervical ganglion, SCG) M/KCNQ currents and excitability. As reported before, subpopulation SCG neurons with distinct firing properties could be classified into tonic, phasic-1, and phasic-2 neurons. NGF inhibited M/KCNQ currents by similar proportion in all three classes of SCG neurons but increased the excitability only significantly in tonic SCG neurons. The effect of NGF on excitability correlated with a smaller M-current density in tonic neurons. The present study indicates that NGF is an M/KCNQ channel modulator and the characteristic modulation of the neuronal excitability by NGF may have important physiological implications.

KCNQ1 and KCNE1 in the IKs Channel Complex Make State-dependent Contacts in their Extracellular Domains

Xu, X., Jiang, M., Hsu, K.-L., Zhang, M., Tseng, G.-N. — 2008-05-26

KCNQ1 and KCNE1 (Q1 and E1) associate to form the slow delayed rectifier I_Ks channels in the heart. A short stretch of eight amino acids at the extracellular end of S1 in Q1 (positions 140–147) harbors six arrhythmia-associated mutations. Some of these mutations affect the Q1 channel function only when coexpressed with E1, suggesting that this Q1 region may engage in the interaction with E1 critical for the I_Ks channel function. Identifying the Q1/E1 contact points here may provide new insights into how the I_Ks channel operates. We focus on Q1 position 145 and E1 positions 40–43. Replacing all native cysteine (Cys) in Q1 and introducing Cys into the above Q1 and E1 positions do not significantly perturb the Q1 channel function or Q1/E1 interactions. Immunoblot experiments on COS-7 cells reveal that Q1 145C can form disulfide bonds with E1 40C and 41C, but not E1 42C or 43C. Correspondingly, voltage clamp experiments in oocytes reveal that Q1 145C coexpressed with E1 40C or E1 41C manifests unique gating behavior and DTT sensitivity. Our data suggest that E1 40C and 41C come close to Q1 145C in the activated and resting states, respectively, to allow disulfide bond formation. These data and those in the literature lead us to propose a structural model for the Q1/E1 channel complex, in which E1 is located between S1, S4, and S6 of three separate Q1 subunits. We propose that E1 is not a passive partner of the Q1 channel, but instead can engage in molecular motions during I_Ks gating.

MEC-2 and MEC-6 in the Caenorhabditis elegans Sensory Mechanotransduction Complex: Auxiliary Subunits that Enable Channel Activity

Brown, A. L., Liao, Z., Goodman, M. B. — 2008-05-26

The ion channel formed by the homologous proteins MEC-4 and MEC-10 forms the core of a sensory mechanotransduction channel in Caenorhabditis elegans. Although the products of other mec genes are key players in the biophysics of transduction, the mechanism by which they contribute to the properties of the channel is unknown. Here, we investigate the role of two auxiliary channel subunits, MEC-2 (stomatin-like) and MEC-6 (paraoxonase-like), by coexpressing them with constitutively active MEC-4/MEC-10 heteromeric channels in Xenopus oocytes. This work extends prior work demonstrating that MEC-2 and MEC-6 synergistically increase macroscopic current. We use single-channel recordings and biochemistry to show that these auxiliary subunits alter function by increasing the number of channels in an active state rather than by dramatically affecting either single-channel properties or surface expression. We also use two-electrode voltage clamp and outside-out macropatch recording to examine the effects of divalent cations and proteases, known regulators of channel family members. Finally, we examine the role of cholesterol binding in the mechanism of MEC-2 action by measuring whole-cell and single-channel currents in MEC-2 mutants deficient in cholesterol binding. We suggest that MEC-2 and MEC-6 play essential roles in modulating both the local membrane environment of MEC-4/MEC-10 channels and the availability of such channels to be gated by force in vivo.

Surface Expression of Epithelial Na Channel Protein in Rat Kidney

Frindt, G., Ergonul, Z., Palmer, L. G. — 2008-05-26

Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes were solubilized and labeled proteins were isolated using neutravidin beads, and surface β and ENaC subunits were assayed by immunoblot. Surface ENaC was assessed by membrane fractionation. Most of the ENaC at the surface was smaller in molecular mass than the full-length subunit, consistent with cleavage of this subunit in the extracellular moiety close to the first transmembrane domains. Insensitivity of the channels to trypsin, measured in principal cells of the cortical collecting duct by whole-cell patch-clamp recording, corroborated this finding. ENaC subunits could be detected at the surface under all physiological conditions. However increasing the levels of aldosterone in the animals by feeding a low-Na diet or infusing them directly with hormone via osmotic minipumps for 1 wk before surface labeling increased the expression of the subunits at the surface by two- to fivefold. Salt repletion of Na-deprived animals for 5 h decreased surface expression. Changes in the surface density of ENaC subunits contribute significantly to the regulation of Na transport in renal cells by mineralocorticoid hormone, but do not fully account for increased channel activity.

Structure and Function of Skeletal Muscle in Zebrafish Early Larvae

Dou, Y., Andersson-Lendahl, M., Arner, A. — 2008-04-28

Zebrafish muscles were examined at an early developmental stage (larvae 5–7 d). Using aluminum clips, preparations (~1.5 mm length, 150 µm diameter) were mounted for force registration and small angle x-ray diffraction. Sarcomeres were oriented mainly in parallel with the preparation long axis. Electrical stimulation elicited fast and reproducible single twitch contractions. Length–force relations showed an optimal sarcomere length of 2.15 µm. x-ray diffraction revealed clear equatorial 1.1/1.0 reflections, showing that myofilaments are predominantly arranged along the preparation long axis. In contrast, reflections from older (2 mo) zebrafish showed two main filament orientations each at an ~25° angle relative to the preparation long axis. Electrical stimulation of larvae muscles increased the 1.1/1.0 intensity ratio, reflecting mass transfer to thin filaments during contraction. The apparent lattice volume was 3.42 x 10^–3 µm³, which is smaller than that of mammalian striated muscle and more similar to that of frog muscles. The relation between force and stimulation frequency showed fusion of responses at a comparatively high frequency (~186 Hz), reflecting a fast muscle phenotype. Inhibition of fast myosin with N-benzyl-p-toluene sulphonamide (BTS) showed that the later phase of the tetanus was less affected than the initial peak. This suggests that, although the main contractile phenotype is fast, slow twitch fibers can contribute to sustained contraction. A fatigue stimulation protocol with repeated 220 ms/186 Hz tetani showed that tetanic force decreased to 50% at a train rate of 0.1 s^–1. In conclusion, zebrafish larvae muscles can be examined in vitro using mechanical and x-ray methods. The muscles and myofilaments are mainly orientated in parallel with the larvae long axis and exhibit a significant fast contractile component. Sustained contractions can also involve a small contribution from slower muscle types.

A Role for DPPX Modulating External TEA Sensitivity of Kv4 Channels

Colinas, O., Perez-Carretero, F. D., Lopez-Lopez, J. R., Perez-Garcia, M. T. — 2008-04-28

Shal-type (Kv4) channels are expressed in a large variety of tissues, where they contribute to transient voltage-dependent K⁺ currents. Kv4 are the molecular correlate of the A-type current of neurons (I_SA), the fast component of I_TO current in the heart, and also of the oxygen-sensitive K⁺ current (K_O2) in rabbit carotid body (CB) chemoreceptor cells. The enormous degree of variability in the physiological properties of Kv4-mediated currents can be attributable to the complexity of their regulation together with the large number of ancillary subunits and scaffolding proteins that associate with Kv4 proteins to modify their trafficking and their kinetic properties. Among those, KChIPs and DPPX proteins have been demonstrated to be integral components of I_SA and I_TO currents, as their coexpression with Kv4 subunits recapitulates the kinetics of native currents. Here, we explore the presence and functional contribution of DPPX to K_O2 currents in rabbit CB chemoreceptor cells by using DPPX functional knockdown with siRNA. Additionally, we investigate if the presence of DPPX endows Kv4 channels with new pharmacological properties, as we have observed anomalous tetraethylammonium (TEA) sensitivity in the native K_O2 currents. DPPX association with Kv4 channels induced an increased TEA sensitivity both in heterologous expression systems and in CB chemoreceptor cells. Moreover, TEA application to Kv4-DPPX heteromultimers leads to marked kinetic effects that could be explained by an augmented closed-state inactivation. Our data suggest that DPPX proteins are integral components of K_O2 currents, and that their association with Kv4 subunits modulate the pharmacological profile of the heteromultimers.

Clearance of Extracellular K+ during Muscle Contraction--Roles of Membrane Transport and Diffusion

Clausen, T. — 2008-04-28

Excitation of muscle often leads to a net loss of cellular K⁺ and a rise in extracellular K⁺ ([ K⁺ ]_o), which in turn inhibits excitability and contractility. It is important, therefore, to determine how this K⁺ is cleared by diffusion into the surroundings or by reaccumulation into the muscle cells. The inhibitory effects of the rise in [K⁺ ]_o may be assessed from the time course of changes in tetanic force in isolated muscles where diffusional clearance of K⁺ is eliminated by removing the incubation medium and allowing the muscles to contract in air. Measurements of tetanic force, endurance, and force recovery showed that in rat soleus and extensor digitorum longus (EDL) muscles there was no significant difference between the performance of muscles contracting in buffer or in air for up to 8 min. Ouabain-induced inhibition of K⁺ clearance via the Na⁺,K⁺ pumps markedly reduced contractile endurance and force recovery in air. Incubation in buffer containing 10 mM K⁺ clearly inhibited force development and endurance, and these effects were considerably reduced by stimulating Na⁺,K⁺ pumps with the β₂-agonist salbutamol. Following 30–60 s of continuous stimulation at 60 Hz, the amount of K⁺ released into the extracellular space was assessed from washout experiments. The release of intracellular K⁺ per pulse was fourfold larger in EDL than in soleus, and in the two muscles, the average [K⁺ ]_o reached 52.4 and 26.0 mM, respectively, appreciably higher than previously detected. In conclusion, prevention of diffusion of K⁺ from the extracellular space of isolated working muscles causes only modest interference with contractile performance. The Na⁺,K⁺ pumps play a major role in the clearance of K⁺ and the maintenance of force. This new information is important for the evaluation of K⁺-induced inhibition in muscles, where diffusional clearance of K⁺ is reduced by tension development sufficient to suppress circulation.

An Extracellular Cu2+ Binding Site in the Voltage Sensor of BK and Shaker Potassium Channels

Ma, Z., Wong, K. Y., Horrigan, F. T. — 2008-04-28

Copper is an essential trace element that may serve as a signaling molecule in the nervous system. Here we show that extracellular Cu²⁺ is a potent inhibitor of BK and Shaker K⁺ channels. At low micromolar concentrations, Cu²⁺ rapidly and reversibly reduces macrosocopic K⁺ conductance (G_K) evoked from mSlo1 BK channels by membrane depolarization. G_K is reduced in a dose-dependent manner with an IC₅₀ and Hill coefficient of ~2 µM and 1.0, respectively. Saturating 100 µM Cu²⁺ shifts the G_K-V relation by +74 mV and reduces G_Kmax by 27% without affecting single channel conductance. However, 100 µM Cu²⁺ fails to inhibit G_K when applied during membrane depolarization, suggesting that Cu²⁺ interacts poorly with the activated channel. Of other transition metal ions tested, only Zn²⁺ and Cd²⁺ had significant effects at 100 µM with IC₅₀s > 0.5 mM, suggesting the binding site is Cu²⁺ selective. Mutation of external Cys or His residues did not alter Cu²⁺ sensitivity. However, four putative Cu²⁺-coordinating residues were identified (D133, Q151, D153, and R207) in transmembrane segments S1, S2, and S4 of the mSlo1 voltage sensor, based on the ability of substitutions at these positions to alter Cu²⁺ and/or Cd²⁺ sensitivity. Consistent with the presence of acidic residues in the binding site, Cu²⁺ sensitivity was reduced at low extracellular pH. The three charged positions in S1, S2, and S4 are highly conserved among voltage-gated channels and could play a general role in metal sensitivity. We demonstrate that Shaker, like mSlo1, is much more sensitive to Cu²⁺ than Zn²⁺ and that sensitivity to these metals is altered by mutating the conserved positions in S1 or S4 or reducing pH. Our results suggest that the voltage sensor forms a state- and pH-dependent, metal-selective binding pocket that may be occupied by Cu²⁺ at physiologically relevant concentrations to inhibit activation of BK and other channels.

Cholesterol Promotes Hemifusion and Pore Widening in Membrane Fusion Induced by Influenza Hemagglutinin

Biswas, S., Yin, S.-R., Blank, P. S., Zimmerberg, J. — 2008-04-28

Cholesterol-specific interactions that affect membrane fusion were tested for using insect cells; cells that have naturally low cholesterol levels (<4 mol %). Sf9 cells were engineered (HAS cells) to express the hemagglutinin (HA) of the influenza virus X-31 strain. Enrichment of HAS cells with cholesterol reduced the delay between triggering and lipid dye transfer between HAS cells and human red blood cells (RBC), indicating that cholesterol facilitates membrane lipid mixing prior to fusion pore opening. Increased cholesterol also increased aqueous content transfer between HAS cells and RBC over a broad range of HA expression levels, suggesting that cholesterol also favors fusion pore expansion. This interpretation was tested using both trans-cell dye diffusion and fusion pore conductivity measurements in cholesterol-enriched cells. The results of this study support the hypothesis that host cell cholesterol acts at two stages in membrane fusion: (1) early, prior to fusion pore opening, and (2) late, during fusion pore expansion.

Calcium Transport Mechanisms of PC12 Cells

Duman, J. G., Chen, L., Hille, B. — 2008-03-31

Many studies of Ca²⁺ signaling use PC12 cells, yet the balance of Ca²⁺ clearance mechanisms in these cells is unknown. We used pharmacological inhibition of Ca²⁺ transporters to characterize Ca²⁺ clearance after depolarizations in both undifferentiated and nerve growth factor-differentiated PC12 cells. Sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA), plasma membrane Ca²⁺ ATPase (PMCA), and Na⁺/Ca²⁺ exchanger (NCX) account for almost all Ca²⁺ clearance in both cell states, with NCX and PMCA making the greatest contributions. Any contribution of mitochondrial uniporters is small. The ATP pool in differentiated cells was much more labile than that of undifferentiated cells in the presence of agents that dissipated mitochondrial proton gradients. Differentiated PC12 cells have a small component of Ca²⁺ clearance possessing pharmacological characteristics consistent with secretory pathway Ca²⁺ ATPase (SPCA), potentially residing on Golgi and/or secretory granules. Undifferentiated and differentiated cells are similar in overall Ca²⁺ transport and in the small transport due to SERCA, but they differ in the fraction of transport by PMCA and NCX. Transport in neurites of differentiated PC12 cells was qualitatively similar to that in the somata, except that the ER stores in neurites sometimes released Ca²⁺ instead of clearing it after depolarization. We formulated a mathematical model to simulate the observed Ca²⁺ clearance and to describe the differences between these undifferentiated and NGF-differentiated states quantitatively. The model required a value for the endogenous Ca²⁺ binding ratio of PC12 cell cytoplasm, which we measured to be 268 ± 85. Our results indicate that Ca²⁺ transport in undifferentiated PC12 cells is quite unlike transport in adrenal chromaffin cells, for which they often are considered models. Transport in both cell states more closely resembles that of sympathetic neurons, for which differentiated PC12 cells often are considered models. Comparison with other cell types shows that different cells emphasize different Ca²⁺ transport mechanisms.

Gap Junction Channels Exhibit Connexin-specific Permeability to Cyclic Nucleotides

2008-03-31

Gap junction channels exhibit connexin dependent biophysical properties, including selective intercellular passage of larger solutes, such as second messengers and siRNA. Here, we report the determination of cyclic nucleotide (cAMP) permeability through gap junction channels composed of Cx43, Cx40, or Cx26 using simultaneous measurements of junctional conductance and intercellular transfer of cAMP. For cAMP detection the recipient cells were transfected with a reporter gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents (I_h) were recorded from the other cell of a pair that expressed SpIH. cAMP diffusion through gap junction channels to the neighboring SpIH-transfected cell resulted in a five to sixfold increase in I_h current over time. Cyclic AMP transfer was observed for homotypic Cx43 channels over a wide range of conductances. However, homotypic Cx40 and homotypic Cx26 exhibited reduced cAMP permeability in comparison to Cx43. The cAMP/K⁺ permeability ratios were 0.18, 0.027, and 0.018 for Cx43, Cx26, and Cx40, respectively. Cx43 channels were ~10 to 7 times more permeable to cAMP than Cx40 or Cx26 (Cx43 > Cx26 ≥ Cx40), suggesting that these channels have distinctly different selectivity for negatively charged larger solutes involved in metabolic/biochemical coupling. These data suggest that Cx43 permeability to cAMP results in a rapid delivery of cAMP from cell to cell in sufficient quantity before degradation by phosphodiesterase to trigger relevant intracellular responses. The data also suggest that the reduced permeability of Cx26 and Cx40 might compromise their ability to deliver cAMP rapidly enough to cause functional changes in a recipient cell.

Luminal Ca2+ Regulation of Single Cardiac Ryanodine Receptors: Insights Provided by Calsequestrin and its Mutants

2008-03-31

The luminal Ca2+ regulation of cardiac ryanodine receptor (RyR2) was explored at the single channel level. The luminal Ca2+ and Mg2+ sensitivity of single CSQ2-stripped and CSQ2-associated RyR2 channels was defined. Action of wild-type CSQ2 and of two mutant CSQ2s (R33Q and L167H) was also compared. Two luminal Ca2+ regulatory mechanism(s) were identified. One is a RyR2-resident mechanism that is CSQ2 independent and does not distinguish between luminal Ca2+ and Mg2+. This mechanism modulates the maximal efficacy of cytosolic Ca2+ activation. The second luminal Ca2+ regulatory mechanism is CSQ2 dependent and distinguishes between luminal Ca2+ and Mg2+. It does not depend on CSQ2 oligomerization or CSQ2 monomer Ca2+ binding affinity. The key Ca2+-sensitive step in this mechanism may be the Ca2+-dependent CSQ2 interaction with triadin. The CSQ2-dependent mechanism alters the cytosolic Ca2+ sensitivity of the channel. The R33Q CSQ2 mutant can participate in luminal RyR2 Ca2+ regulation but less effectively than wild-type (WT) CSQ2. CSQ2-L167H does not participate in luminal RyR2 Ca2+ regulation. The disparate actions of these two catecholaminergic polymorphic ventricular tachycardia (CPVT)–linked mutants implies that either alteration or elimination of CSQ2-dependent luminal RyR2 regulation can generate the CPVT phenotype. We propose that the RyR2-resident, CSQ2-independent luminal Ca2+ mechanism may assure that all channels respond robustly to large (>5 µM) local cytosolic Ca2+ stimuli, whereas the CSQ2-dependent mechanism may help close RyR2 channels after luminal Ca2+ falls below ~0.5 mM.

Calcium-dependent Inactivation Terminates Calcium Release in Skeletal Muscle of Amphibians

Rios, E., Zhou, J., Brum, G., Launikonis, B. S., Stern, M. D. — 2008-03-31

In skeletal muscle of amphibians, the cell-wide cytosolic release of calcium that enables contraction in response to an action potential appears to be built of Ca²⁺ sparks. The mechanism that rapidly terminates this release was investigated by studying the termination of Ca²⁺ release underlying sparks. In groups of thousands of sparks occurring spontaneously in membrane-permeabilized frog muscle cells a complex relationship was found between amplitude a and rise time T, which in sparks corresponds to the active time of the underlying Ca²⁺ release. This relationship included a range of T where a paradoxically decreased with increasing T. Three different methods were used to estimate Ca²⁺ release flux in groups of sparks of different T. Using every method, it was found that T and flux were inversely correlated, roughly inversely proportional. A simple model in which release sources were inactivated by cytosolic Ca²⁺ was able to explain the relationship. The predictive value of the model, evaluated by analyzing the variance of spark amplitude, was found to be high when allowance was made for the out-of-focus error contribution to the total variance. This contribution was estimated using a theory of confocal scanning (Ríos, E., N. Shirokova, W.G. Kirsch, G. Pizarro, M.D. Stern, H. Cheng, and A. González. Biophys. J. 2001. 80:169–183), which was confirmed in the present work by simulated line scanning of simulated sparks. Considering these results and other available evidence it is concluded that Ca²⁺-dependent inactivation, or CDI, provides the crucial mechanism for termination of sparks and cell-wide Ca²⁺ release in amphibians. Given the similarities in kinetics of release termination observed in cell-averaged records of amphibian and mammalian muscle, and in spite of differences in activation mechanisms, CDI is likely to play a central role in mammals as well. Trivially, an inverse proportionality between release flux and duration, in sparks or in global release of skeletal muscle, maintains constancy of the amount of released Ca²⁺.

Roles of GRK and PDE4 Activities in the Regulation of {beta}2 Adrenergic Signaling

Xin, W., Tran, T. M., Richter, W., Clark, R. B., Rich, T. C. — 2008-03-31

An important focus in cell biology is understanding how different feedback mechanisms regulate G protein–coupled receptor systems. Toward this end we investigated the regulation of endogenous β₂ adrenergic receptors (β2ARs) and phosphodiesterases (PDEs) by measuring cAMP signals in single HEK-293 cells. We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels. This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 µM isoproterenol triggered transient increases in cAMP levels near the plasma membrane. Pretreatment of cells with 10 µM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 µM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 µM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included. Taken together, these data indicate that either GRK-mediated desensitization of β2ARs or PKA-mediated stimulation of PDE4 activity is sufficient to cause declines in cAMP signals. In addition, the data indicate that GRK-mediated desensitization is primarily responsible for a sustained suppression of β2AR signaling. To better understand the interplay between receptor desensitization and PDE4 activity in controlling cAMP signals, we developed a mathematical model of this system. Simulations of cAMP signals using this model are consistent with the experimental data and demonstrate the importance of receptor levels, receptor desensitization, basal adenylyl cyclase activity, and regulation of PDE activity in controlling cAMP signals, and hence, on the overall sensitivity of the system.

Tl+-induced {micro}s Gating of Current Indicates Instability of the MaxiK Selectivity Filter as Caused by Ion/Pore Interaction

Schroeder, I., Hansen, U.-P. — 2008-03-31

Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed at high temporal resolution (50-kHz filter) in asymmetrical solutions containing 0, 25, 50, or 150 mM Tl⁺ on the luminal or cytosolic side with [K⁺] + [Tl⁺] = 150 mM and 150 mM K⁺ on the other side. Outward current in the presence of cytosolic Tl⁺ did not show fast gating behavior that was significantly different from that in the absence of Tl⁺. With luminal Tl⁺ and at membrane potentials more negative than –40 mV, the single-channel current showed a negative slope resistance concomitantly with a flickery block, resulting in an artificially reduced apparent single-channel current I_app. The analysis of the amplitude histograms by β distributions enabled the estimation of the true single-channel current and the determination of the rate constants of a simple two-state O-C Markov model for the gating in the bursts. The voltage dependence of the gating ratio R = I_true/I_app = (k_CO + k_OC)/k_CO could be described by exponential functions with different characteristic voltages above or below 50 mM Tl⁺. The true single-channel current I_true decreased with Tl⁺ concentrations up to 50 mM and stayed constant thereafter. Different models were considered. The most likely ones related the exponential increase of the gating ratio to ion depletion at the luminal side of the selectivity filter, whereas the influence of [Tl⁺] on the characteristic voltage of these exponential functions and of the value of I_true were determined by [Tl⁺] at the inner side of the selectivity filter or in the cavity.

Chloride Homeostasis in Saccharomyces cerevisiae: High Affinity Influx, V-ATPase-dependent Sequestration, and Identification of a Candidate Cl- Sensor

Jennings, M. L., Cui, J. — 2008-03-31

Chloride homeostasis in Saccharomyces cerevisiae has been characterized with the goal of identifying new Cl^– transport and regulatory pathways. Steady-state cellular Cl^– contents (~0.2 mEq/liter cell water) differ by less than threefold in yeast grown in media containing 0.003–5 mM Cl^–. Therefore, yeast have a potent mechanism for maintaining constant cellular Cl^– over a wide range of extracellular Cl^–. The cell water:medium [Cl^–] ratio is >20 in media containing 0.01 mM Cl^– and results in part from sequestration of Cl^– in organelles, as shown by the effect of deleting genes involved in vacuolar acidification. Organellar sequestration cannot account entirely for the Cl^– accumulation, however, because the cell water:medium [Cl^–] ratio in low Cl^– medium is ~10 at extracellular pH 4.0 even in vma1 yeast, which lack the vacuolar H⁺-ATPase. Cellular Cl^– accumulation is ATP dependent in both wild type and vma1 strains. The initial ³⁶Cl^– influx is a saturable function of extracellular [³⁶Cl^–] with K_1/2 of 0.02 mM at pH 4.0 and >0.2 mM at pH 7, indicating the presence of a high affinity Cl^– transporter in the plasma membrane. The transporter can exchange ³⁶Cl^– for either Cl^– or Br^– far more rapidly than SO₄⁼, phosphate, formate, HCO₃^–, or NO₃^–. High affinity Cl^– influx is not affected by deletion of any of several genes for possible Cl^– transporters. The high affinity Cl^– transporter is activated over a period of ~45 min after shifting cells from high-Cl^– to low-Cl^– media. Deletion of ORF YHL008c (formate-nitrite transporter family) strongly reduces the rate of activation of the flux. Therefore, Yhl008cp may be part of a Cl^–-sensing mechanism that activates the high affinity transporter in a low Cl^– medium. This is the first example of a biological system that can regulate cellular Cl^– at concentrations far below 1 mM.