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The Legacy of Olaf Sparre Andersen and Future Directions of the Journal of General Physiology

Pugh, E. N. — 2008-06-30

Abstracts of Papers at the Sixty-Second Annual Meeting of the Society of General Physiologists

2008-06-30

PIP2 PIP2 Hooray for Maxi K+

Rittenhouse, A. R. — 2008-06-30

Fusion Gains Independence

Lindau, M. — 2008-06-30

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.

INDEX TO AUTHORS OF ABSTRACTS

2008-06-30

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.

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.

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.

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.

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.

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.

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.