The Journal of General Physiology Articles

The Ca2+ channel {beta} subunit determines whether stimulation of Gq-coupled receptors enhances or inhibits N current

Mon, 26 Oct 2009 10:01:46 PDT

In superior cervical ganglion (SCG) neurons, stimulation of M₁ receptors (M₁Rs) produces a distinct pattern of modulation of N-type calcium (N-) channel activity, enhancing currents elicited with negative test potentials and inhibiting currents elicited with positive test potentials. Exogenously applied arachidonic acid (AA) reproduces this profile of modulation, suggesting AA functions as a downstream messenger of M₁Rs. In addition, techniques that diminish AA's concentration during M₁R stimulation minimize N-current modulation. However, other studies suggest depletion of phosphatidylinositol-4,5-bisphosphate during M₁R stimulation suffices to elicit modulation. In this study, we used an expression system to examine the physiological mechanisms regulating modulation. We found the β subunit (Ca_Vβ) acts as a molecular switch regulating whether modulation results in enhancement or inhibition. In human embryonic kidney 293 cells, stimulation of M₁Rs or neurokinin-1 receptors (NK-1Rs) inhibited activity of N channels formed by Ca_V2.2 and coexpressed with Ca_Vβ1b, Ca_Vβ3, or Ca_Vβ4 but enhanced activity of N channels containing Ca_Vβ2a. Exogenously applied AA produced the same pattern of modulation. Coexpression of Ca_Vβ2a, Ca_Vβ3, and Ca_Vβ4 recapitulated the modulatory response previously seen in SCG neurons, implying heterogeneous association of Ca_Vβ with Ca_V2.2. Further experiments with mutated, chimeric Ca_Vβ subunits and free palmitic acid revealed that palmitoylation of Ca_Vβ2a is essential for loss of inhibition. The data presented here fit a model in which Ca_Vβ2a blocks inhibition, thus unmasking enhancement. Our discovery that the presence or absence of palmitoylated Ca_Vβ2a toggles M₁R- or NK-1R–mediated modulation of N current between enhancement and inhibition identifies a novel role for palmitoylation. Moreover, these findings predict that at synapses, modulation of N-channel activity by M₁Rs or NK-1Rs will fluctuate between enhancement and inhibition based on the presence of palmitoylated Ca_Vβ2a.

Orientation of palmitoylated CaV{beta}2a relative to CaV2.2 is critical for slow pathway modulation of N-type Ca2+ current by tachykinin receptor activation

Mitra-Ganguli, T., Vitko, I., Perez-Reyes, E., Rittenhouse, A. R. — Mon, 26 Oct 2009 10:01:46 PDT

The G_q-coupled tachykinin receptor (neurokinin-1 receptor [NK-1R]) modulates N-type Ca²⁺ channel (Ca_V2.2 or N channel) activity at two distinct sites by a pathway involving a lipid metabolite, most likely arachidonic acid (AA). In another study published in this issue (Heneghan et al. 2009. J. Gen Physiol. doi:10.1085/jgp.200910203), we found that the form of modulation observed depends on which Ca_Vβ is coexpressed with Ca_V2.2. When palmitoylated Ca_Vβ2a is coexpressed, activation of NK-1Rs by substance P (SP) enhances N current. In contrast, when Ca_Vβ3 is coexpressed, SP inhibits N current. However, exogenously applied palmitic acid minimizes this inhibition. These findings suggested that the palmitoyl groups of Ca_Vβ2a may occupy an inhibitory site on Ca_V2.2 or prevent AA from interacting with that site, thereby minimizing inhibition. If so, changing the orientation of Ca_Vβ2a relative to Ca_V2.2 may displace the palmitoyl groups and prevent them from antagonizing AA's actions, thereby allowing inhibition even in the presence of Ca_Vβ2a. In this study, we tested this hypothesis by deleting one (Bdel1) or two (Bdel2) amino acids proximal to the interacting domain (AID) of Ca_V2.2's I–II linker. Ca_Vβs bind tightly to the AID, whereas the rigid region proximal to the AID is thought to couple Ca_Vβ's movements to Ca_V2.2 gating. Although Bdel1/β2a currents exhibited more variable enhancement by SP, Bdel2/β2a current enhancement was lost at all voltages. Instead, inhibition was observed that matched the profile of N-current inhibition from Ca_V2.2 coexpressed with Ca_Vβ3. Moreover, adding back exogenous palmitic acid minimized inhibition of Bdel2/β2a currents, suggesting that when palmitoylated Ca_Vβ2a is sufficiently displaced, endogenously released AA can bind to the inhibitory site. These findings support our previous hypothesis that Ca_Vβ2a's palmitoyl groups directly interact with an inhibitory site on Ca_V2.2 to block N-current inhibition by SP.

Activation and desensitization of the olfactory cAMP-gated transduction channel: identification of functional modules

Waldeck, C., Vocke, K., Ungerer, N., Frings, S., Mohrlen, F. — Mon, 26 Oct 2009 10:01:46 PDT

Olfactory receptor neurons respond to odor stimulation with a receptor potential that results from the successive activation of cyclic AMP (cAMP)-gated, Ca²⁺-permeable channels and Ca²⁺-activated chloride channels. The cAMP-gated channels open at micromolar concentrations of their ligand and are subject to a Ca²⁺-dependent feedback inhibition by calmodulin. Attempts to understand the operation of these channels have been hampered by the fact that the channel protein is composed of three different subunits, CNGA2, CNGA4, and CNGB1b. Here, we explore the individual role that each subunit plays in the gating process. Using site-directed mutagenesis and patch clamp analysis, we identify three functional modules that govern channel operation: a module that opens the channel, a module that stabilizes the open state at low cAMP concentrations, and a module that mediates rapid Ca²⁺-dependent feedback inhibition. Each subunit could be assigned to one of these functions that, together, define the gating logic of the olfactory transduction channel.

Closed-channel block of BK potassium channels by bbTBA requires partial activation

Tang, Q.-Y., Zeng, X.-H., Lingle, C. J. — Mon, 26 Oct 2009 10:01:46 PDT

Blockade of large-conductance Ca²⁺-activated K⁺ (BK) channels by the bulky quaternary ammonium compound, N-(4-[benzoyl]benzyl)-N,N,N-tributylammonium (bbTBA), exhibits features consistent with blockade of both closed and open states. Here, we examine block of closed BK channels by bbTBA and how it may differ from block of open channels. Although our observations generally confirm earlier results, we describe three observations that are inconsistent with a model in which closed and open channels are equally accessible to blockade by bbTBA. First, block by bbTBA exhibits Ca²⁺-dependent features that are inconsistent with strictly state-independent block. Second, the steady-state voltage dependence of bbTBA block at negative potentials shows that any block of completely closed states either does not occur or is completely voltage independent. Third, determination of the fractional unblock by bbTBA at either low or high Ca²⁺ reveals deviations from a model in which open- and closed-state block is identical. The results support the view that bbTBA blockade of fully closed channels does not occur. We imagine two general types of explanation. First, a stronger voltage dependence of closed-channel block may minimize the contribution of closed-channel block at negative potentials. Second, voltage-dependent conformational changes among closed-channel states may permit block by bbTBA. The analysis supports the latter view, suggesting that bbTBA blockade of fully closed channels does not occur, but the ability of bbTBA to block a closed channel requires movement of one or more voltage sensors. Models in which block is coupled to voltage sensor movement can qualitatively account for (1) the ability of open-channel block to better fit block of conductance–voltage curves at high Ca²⁺; (2) the voltage dependence of fractional availability; and (3) the fractional unblock at different open probabilities. BK channels appear to undergo voltage-dependent conformational changes among closed states that are permissive for bbTBA block.

Affinity for phosphatidylinositol 4,5-bisphosphate determines muscarinic agonist sensitivity of Kv7 K+ channels

Hernandez, C. C., Falkenburger, B., Shapiro, M. S. — Mon, 26 Oct 2009 10:01:46 PDT

Kv7 K⁺-channel subunits differ in their apparent affinity for PIP₂ and are differentially expressed in nerve, muscle, and epithelia in accord with their physiological roles in those tissues. To investigate how PIP₂ affinity affects the response to physiological stimuli such as receptor stimulation, we exposed homomeric and heteromeric Kv7.2, 7.3, and 7.4 channels to a range of concentrations of the muscarinic receptor agonist oxotremorine-M (oxo-M) in a heterologous expression system. Activation of M₁ receptors by oxo-M leads to PIP₂ depletion through G_q and phospholipase C (PLC). Chinese hamster ovary cells were transiently transfected with Kv7 subunits and M₁ receptors and studied under perforated-patch voltage clamp. For Kv7.2/7.3 heteromers, the EC₅₀ for current suppression was 0.44 ± 0.08 µM, and the maximal inhibition (Inhib_max) was 74 ± 3% (n = 5–7). When tonic PIP₂ abundance was increased by overexpression of PIP 5-kinase, the EC₅₀ was shifted threefold to the right (1.2 ± 0.1 µM), but without a significant change in Inhib_max (73 ± 4%, n = 5). To investigate the muscarinic sensitivity of Kv7.3 homomers, we used the A315T pore mutant (Kv7.3^T) that increases whole-cell currents by 30-fold without any change in apparent PIP₂ affinity. Kv7.3^T currents had a slightly right-shifted EC₅₀ as compared with Kv7.2/7.3 heteromers (1.0 ± 0.8 µM) and a strongly reduced Inhib_max (39 ± 3%). In contrast, the dose–response curve of homomeric Kv7.4 channels was shifted considerably to the left (66 ± 8 nM), and Inhib_max was slightly increased (81 ± 6%, n = 3–4). We then studied several Kv7.2 mutants with altered apparent affinities for PIP₂ by coexpressing them with Kv7.3^T subunits to boost current amplitudes. For the lower affinity (Kv7.2 (R463Q)/Kv7.3^T) or higher affinity (Kv7.2 (R463E)/Kv7.3^T) channels, the EC₅₀ and Inhib_max were similar to Kv7.4 or Kv7.3^T homomers (0.12 ± 0.08 µM and 79 ± 6% [n = 3–4] and 0.58 ± 0.07 µM and 27 ± 3% [n = 3–4], respectively). The very low-affinity Kv7.2 (R452E, R459E, and R461E) triple mutant was also coexpressed with Kv7.3^T. The resulting heteromer displayed a very low EC₅₀ for inhibition (32 ± 8 nM) and a slightly increased Inhib_max (83 ± 3%, n = 3–4). We then constructed a cellular model that incorporates PLC activation by oxo-M, PIP₂ hydrolysis, PIP₂ binding to Kv7-channel subunits, and K⁺ current through Kv7 tetramers. We were able to fully reproduce our data and extract a consistent set of PIP₂ affinities.

Suppression of Ca2+ syntillas increases spontaneous exocytosis in mouse adrenal chromaffin cells

Mon, 28 Sep 2009 10:01:55 PDT

A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca²⁺] in the vicinity of plasmalemmal Ca²⁺ channels due to Ca²⁺ influx elicits exocytosis. Here, we examine the effect on spontaneous exocytosis of a rise in focal cytosolic [Ca²⁺] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca²⁺ syntillas. Ca²⁺ syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. (scintilla, Latin for spark; found in nerve terminals, normally synaptic structures.) We have also observed Ca²⁺ syntillas in mouse adrenal chromaffin cells. Here, we examine the effect of Ca²⁺ syntillas on exocytosis in chromaffin cells. In such a study on elicited exocytosis, there are two sources of Ca²⁺: one due to influx from the cell exterior through voltage-gated Ca²⁺ channels, and that due to release from intracellular stores. To eliminate complications arising from Ca²⁺ influx, we have examined spontaneous exocytosis where influx is not activated. We report here that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case, release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine, after which syntillas were decreased. We conclude that Ca²⁺ syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas.

EF hands at the N-lobe of calmodulin are required for both SK channel gating and stable SK-calmodulin interaction

Li, W., Halling, D. B., Hall, A. W., Aldrich, R. W. — Mon, 28 Sep 2009 10:01:56 PDT

Small conductance calcium-activated potassium (SK) channels respond to intracellular Ca²⁺ via constitutively associated calmodulin (CaM). Previous studies have proposed a modular design for the interaction between CaM and SK channels. The C-lobe and the linker of CaM are thought to regulate the constitutive binding, whereas the N-lobe binds Ca²⁺ and gates SK channels. However, we found that coexpression of mutant CaM (E/Q) where the N-lobe has only one functional EF hand leads to rapid rundown of SK channel activity, which can be recovered with exogenously applied wild-type (WT), but not mutant, CaM. Our results suggest that the mutation at the N-lobe EF hand disrupts the stable interaction between CaM and SK channel subunits, such that mutant CaM dissociates from the channel complex when the inside of the membrane is exposed to CaM-free solution. The disruption of the stable interaction does not directly result from the loss of Ca²⁺-binding capacity because SK channels and WT CaM can stably interact in the absence of Ca²⁺. These findings question a previous conclusion that CaM where the N-lobe has only one functional EF hand can stably support the gating of SK channels. They cannot be explained by the current model of modular interaction between CaM and SK channels, and they imply a role for N-lobe EF hand residues in binding to the channel subunits. Additionally, we found that a potent enhancer for SK channels, 3-oxime-6,7-dichloro-1H-indole-2,3-dione (NS309), enables the recovery of channel activity with CaM (E/Q), suggesting that NS309 stabilizes the interaction between CaM and SK channels. CaM (E/Q) can regulate Ca²⁺-dependent gating of SK channels in the presence of NS309, but with a lower apparent Ca²⁺ affinity than WT CaM.

Inhibition of KCa2.2 and KCa2.3 channel currents by protonation of outer pore histidine residues

Goodchild, S. J., Lamy, C., Seutin, V., Marrion, N. V. — Mon, 28 Sep 2009 10:01:56 PDT

Ion channels are often modulated by changes in extracellular pH, with most examples resulting from shifts in the ionization state of histidine residue(s) in the channel pore. The application of acidic extracellular solution inhibited expressed K_Ca2.2 (SK2) and K_Ca2.3 (SK3) channel currents, with K_Ca2.3 (pIC₅₀ of ~6.8) being approximately fourfold more sensitive than K_Ca2.2 (pIC₅₀ of ~6.2). Inhibition was found to be voltage dependent, resulting from a shift in the affinity for the rectifying intracellular divalent cation(s) at the inner mouth of the selectivity filter. The inhibition by extracellular protons resulted from a reduction in the single-channel conductance, without significant changes in open-state kinetics or open probability. K_Ca2.2 and K_Ca2.3 subunits both possess a histidine residue in their outer pore region between the transmembrane S5 segment and the pore helix, with K_Ca2.3 also exhibiting an additional histidine residue between the selectivity filter and S6. Mutagenesis revealed that the outer pore histidine common to both channels was critical for inhibition. The greater sensitivity of K_Ca2.3 currents to protons arose from the additional histidine residue in the pore, which was more proximal to the conduction pathway and in the electrostatic vicinity of the ion conduction pathway. The decrease of channel conductance by extracellular protons was mimicked by mutation of the outer pore histidine in K_Ca2.2 to an asparagine residue. These data suggest that local interactions involving the outer turret histidine residues are crucial to enable high conductance openings, with protonation inhibiting current by changing pore shape.

Regulation of ClC-1 and KATP channels in action potential-firing fast-twitch muscle fibers

Pedersen, T. H., de Paoli, F. V., Flatman, J. A., Nielsen, O. B. — Mon, 28 Sep 2009 10:01:56 PDT

Action potential (AP) excitation requires a transient dominance of depolarizing membrane currents over the repolarizing membrane currents that stabilize the resting membrane potential. Such stabilizing currents, in turn, depend on passive membrane conductance (G_m), which in skeletal muscle fibers covers membrane conductances for K⁺ (G_K) and Cl^– (G_Cl). Myotonic disorders and studies with metabolically poisoned muscle have revealed capacities of G_K and G_Cl to inversely interfere with muscle excitability. However, whether regulation of G_K and G_Cl occur in AP-firing muscle under normal physiological conditions is unknown. This study establishes a technique that allows the determination of G_Cl and G_K with a temporal resolution of seconds in AP-firing muscle fibers. With this approach, we have identified and quantified a biphasic regulation of G_m in active fast-twitch extensor digitorum longus fibers of the rat. Thus, at the onset of AP firing, a reduction in G_Cl of ~70% caused G_m to decline by ~55% in a manner that is well described by a single exponential function characterized by a time constant of ~200 APs (phase 1). When stimulation was continued beyond ~1,800 APs, synchronized elevations in G_K (~14-fold) and G_Cl (~3-fold) caused G_m to rise sigmoidally to ~400% of its level before AP firing (phase 2). Phase 2 was often associated with a failure to excite APs. When AP firing was ceased during phase 2, G_m recovered to its level before AP firing in ~1 min. Experiments with glibenclamide (K_ATP channel inhibitor) and 9-anthracene carboxylic acid (ClC-1 Cl^– channel inhibitor) revealed that the decreased G_m during phase 1 reflected ClC-1 channel inhibition, whereas the massively elevated G_m during phase 2 reflected synchronized openings of ClC-1 and K_ATP channels. In conclusion, G_Cl and G_K are acutely regulated in AP-firing fast-twitch muscle fibers. Such regulation may contribute to the physiological control of excitability in active muscle.

Comparison of regulated passive membrane conductance in action potential-firing fast- and slow-twitch muscle

Mon, 28 Sep 2009 10:01:56 PDT

In several pathological and experimental conditions, the passive membrane conductance of muscle fibers (G_m) and their excitability are inversely related. Despite this capacity of G_m to determine muscle excitability, its regulation in active muscle fibers is largely unexplored. In this issue, our previous study (Pedersen et al. 2009. J. Gen. Physiol. doi:10.1085/jgp.200910291) established a technique with which biphasic regulation of G_m in action potential (AP)-firing fast-twitch fibers of rat extensor digitorum longus muscles was identified and characterized with temporal resolution of seconds. This showed that AP firing initially reduced G_m via ClC-1 channel inhibition but after ~1,800 APs, G_m rose substantially, causing AP excitation failure. This late increase of G_m reflected activation of ClC-1 and K_ATP channels. The present study has explored regulation of G_m in AP-firing slow-twitch fibers of soleus muscle and compared it to G_m dynamics in fast-twitch fibers. It further explored aspects of the cellular signaling that conveyed regulation of G_m in AP-firing fibers. Thus, in both fiber types, AP firing first triggered protein kinase C (PKC)-dependent ClC-1 channel inhibition that reduced G_m by ~50%. Experiments with dantrolene showed that AP-triggered SR Ca²⁺ release activated this PKC-mediated ClC-1 channel inhibition that was associated with reduced rheobase current and improved function of depolarized muscles, indicating that the reduced G_m enhanced muscle fiber excitability. In fast-twitch fibers, the late rise in G_m was accelerated by glucose-free conditions, whereas it was postponed when intermittent resting periods were introduced during AP firing. Remarkably, elevation of G_m was never encountered in AP-firing slow-twitch fibers, even after 15,000 APs. These observations implicate metabolic depression in the elevation of G_m in AP-firing fast-twitch fibers. It is concluded that regulation of G_m is a general phenomenon in AP-firing muscle, and that differences in G_m regulation may contribute to the different phenotypes of fast- and slow-twitch muscle.

Trabeculae carneae as models of the ventricular walls: implications for the delivery of oxygen

Mon, 28 Sep 2009 10:01:56 PDT

Trabeculae carneae are the smallest naturally arising collections of linearly arranged myocytes in the heart. They are the preparation of choice for studies of function of intact myocardium in vitro. In vivo, trabeculae are unique in receiving oxygen from two independent sources: the coronary circulation and the surrounding ventricular blood. Because oxygen partial pressure (PO₂) in the coronary arterioles is identical in specimens from both ventricles, whereas that of ventricular blood is 2.5-fold higher in the left ventricle than in the right ventricle, trabeculae represent a "natural laboratory" in which to examine the influence of "extravascular" PO₂ on the extent of capillarization of myocardial tissue. We exploit this advantage to test four hypotheses. (1) In trabeculae from either ventricle, a peripheral annulus of cells is devoid of capillaries. (2) Hence, sufficiently small trabeculae from either ventricle are totally devoid of capillaries. (3) The capillary-to-myocyte ratios in specimens from either ventricle are identical to those of their respective walls. (4) Capillary-to-myocyte ratios are comparable in specimens from either ventricle, reflecting equivalent energy demands in vivo, driven by identical contractile frequencies and comparable wall stresses. We applied confocal fluorescent imaging to trabeculae in cross section, subsequently using semi-automated segmentation techniques to distinguish capillaries from myocytes. We quantified the capillary-to-myocyte ratios of trabeculae from both ventricles and compared them to those determined for the ventricular free walls and septum. Quantitative interpretation was furthered by mathematical modeling, using both the classical solution to the diffusion equation for elliptical cross sections, and a novel approach applicable to cross sections of arbitrary shape containing arbitrary disposition of capillaries and non-respiring collagen cords.

On the functional use of the membrane compartmentalized pool of ATP by the Na+ and Ca++ pumps in human red blood cell ghosts

Hoffman, J. F., Dodson, A., Proverbio, F. — Mon, 28 Sep 2009 10:01:56 PDT

Previous evidence established that a sequestered form of adenosine triphosphate (ATP pools) resides in the membrane/cytoskeletal complex of red cell porous ghosts. Here, we further characterize the roles these ATP pools can perform in the operation of the membrane's Na⁺ and Ca²⁺ pumps. The formation of the Na⁺- and Ca²⁺-dependent phosphointermediates of both types of pumps (E_Na-P and E_Ca-P) that conventionally can be labeled with trace amounts of [-³P]ATP cannot occur when the pools contain unlabeled ATP, presumably because of dilution of the [-³P]ATP in the pool. Running the pumps forward with either Na⁺ or Ca²⁺ removes pool ATP and allows the normal formation of labeled E_Na-P or E_Ca-P, indicating that both types of pumps can share the same pools of ATP. We also show that the halftime for loading the pools with bulk ATP is 10–15 minutes. We observed that when unlabeled "caged ATP" is entrapped in the membrane pools, it is inactive until nascent ATP is photoreleased, thereby blocking the labeled formation of E_Na-P. We also demonstrate that ATP generated by the membrane-bound pyruvate kinase fills the membrane pools. Other results show that pool ATP alone, like bulk ATP, can promote the binding of ouabain to the membrane. In addition, we found that pool ATP alone functions together with bulk Na⁺ (without Mg²⁺) to release prebound ouabain. Curiously, ouabain was found to block bulk ATP from entering the pools. Finally, we show, with red cell inside-outside vesicles, that pool ATP alone supports the uptake of ⁴⁵Ca by the Ca²⁺ pump, analogous to the Na⁺ pump uptake of ²²Na in this circumstance. Although the membrane locus of the ATP pools within the membrane/cytoskeletal complex is unknown, it appears that pool ATP functions as the proximate energy source for the Na⁺ and Ca²⁺ pumps.

Metabolic constraints on the recovery of sensitivity after visual pigment bleaching in retinal rods

Miyagishima, K. J., Cornwall, M. C., Sampath, A. P. — Mon, 31 Aug 2009 12:46:13 PDT

The shutoff of active intermediates in the phototransduction cascade and the reconstitution of the visual pigment play key roles in the recovery of sensitivity after the exposure to bright light in both rod and cone photoreceptors. Physiological evidence from bleached salamander rods suggests this recovery of sensitivity occurs faster at the outer segment base compared with the tip. Microfluorometric measurements of similarly bleached salamander rods demonstrate that the reduction of all-trans retinal to all-trans retinol also occurs more rapidly at the outer segment base than at the tip. The experiments reported here were designed to test the hypothesis that these two phenomena are linked, e.g., that slowed recovery of sensitivity at the tip of outer segments is rate limited by the reduction of all-trans retinal and results from a shortage of cytosolic nicotinamide adenine dinucleotide phosphate (NADPH), the reducing agent for all-trans retinal reduction. Extracellular measurements of membrane current and sensitivity were made from isolated salamander rods under dark-adapted and bleached conditions while intracellular NADPH concentration was varied by dialysis from a micropipette attached to the inner segment. Sensitivity at the base and tip of the outer segment was assessed before and after bleaching. After exposure to a light that photoactivates 50% of the visual pigment, rods were completely insensitive for nearly 10 minutes, after which the base recovered sensitivity and responsiveness with a time constant of ~200 seconds, but tip sensitivity recovered more slowly with a time constant of ~680 seconds. Dialysis of 5 mM NADPH into the rod promoted an earlier recovery and eliminated the previously observed tip/base difference. Dialysis of 1.66 mM NADPH failed to eliminate the tip/base recovery difference, suggesting the steady-state NADPH concentration in rods is ~1 mM. These results indicate the inner segment is the primary source of reducing equivalents after pigment bleaching, with the reduction of all-trans retinal to all-trans retinol playing a key step in the recovery of sensitivity.

Prolonged calcium influx after termination of light-induced calcium release in invertebrate photoreceptors

del Pilar Gomez, M., Nasi, E. — Mon, 31 Aug 2009 12:46:13 PDT

In microvillar photoreceptors, light stimulates the phospholipase C cascade and triggers an elevation of cytosolic Ca²⁺ that is essential for the regulation of both visual excitation and sensory adaptation. In some organisms, influx through light-activated ion channels contributes to the Ca²⁺ increase. In contrast, in other species, such as Lima, Ca²⁺ is initially only released from an intracellular pool, as the light-sensitive conductance is negligibly permeable to calcium ions. As a consequence, coping with sustained stimulation poses a challenge, requiring an alternative pathway for further calcium mobilization. We observed that after bright or prolonged illumination, the receptor potential of Lima photoreceptors is followed by the gradual development of an after-depolarization that decays in 1–4 minutes. Under voltage clamp, a graded, slow inward current (I_slow) can be reproducibly elicited by flashes that saturate the photocurrent, and can reach a peak amplitude in excess of 200 pA. I_slow obtains after replacing extracellular Na⁺ with Li⁺, guanidinium, or N-methyl-d-glucamine, indicating that it does not reflect the activation of an electrogenic Na/Ca exchange mechanism. An increase in membrane conductance accompanies the slow current. I_slow is impervious to anion replacements and can be measured with extracellular Ca²⁺ as the sole permeant species; Ba can substitute for Ca²⁺ but Mg²⁺ cannot. A persistent Ca²⁺ elevation parallels I_slow, when no further internal release takes place. Thus, this slow current could contribute to sustained Ca²⁺ mobilization and the concomitant regulation of the phototransduction machinery. Although reminiscent of the classical store depletion–operated calcium influx described in other cells, I_slow appears to diverge in some significant aspects, such as its large size and insensitivity to SKF96365 and lanthanum; therefore, it may reflect an alternative mechanism for prolonged increase of cytosolic calcium in photoreceptors.

Temperature dependence of proton permeation through a voltage-gated proton channel

Kuno, M., Ando, H., Morihata, H., Sakai, H., Mori, H., Sawada, M., Oiki, S. — Mon, 31 Aug 2009 12:46:13 PDT

Voltage-gated proton channels are found in many different types of cells, where they facilitate proton movement through the membrane. The mechanism of proton permeation through the channel is an issue of long-term interest, but it remains an open question. To address this issue, we examined the temperature dependence of proton permeation. Under whole cell recordings, rapid temperature changes within a few milliseconds were imposed. This method allowed for the measurement of current amplitudes immediately before and after a temperature jump, from which the ratios of these currents (I_ratio) were determined. The use of I_ratio for evaluating the temperature dependence minimized the contributions of factors other than permeation. Temperature jumps of various degrees (T, –15 to 15°C) were applied over a wide temperature range (4–49°C), and the Q₁₀s for the proton currents were evaluated from the I_ratios. Q₁₀ exhibited a high temperature dependence, varying from 2.2 at 10°C to 1.3 at 40°C. This implies that processes with different temperature dependencies underlie the observed Q₁₀. A novel resistivity pulse method revealed that the access resistance with its low temperature dependence predominated in high temperature ranges. The measured temperature dependence of Q₁₀ was decomposed into Q₁₀ of the channel and of the access resistances. Finally, the Q₁₀ for proton permeation through the voltage-gated proton channel itself was calculated and found to vary from 2.8 at 5°C to 2.2 at 45°C, as expected for an activation enthalpy of 64 kJ/mol. The thermodynamic features for proton permeation through proton-selective channels were discussed for the underlying mechanism.

Distinct subdomains of the KCNQ1 S6 segment determine channel modulation by different KCNE subunits

Mon, 31 Aug 2009 12:46:13 PDT

Modulation of voltage-gated potassium (K_V) channels by the KCNE family of single transmembrane proteins has physiological and pathophysiological importance. All five KCNE proteins (KCNE1–KCNE5) have been demonstrated to modulate heterologously expressed KCNQ1 (K_V7.1) with diverse effects, making this channel a valuable experimental platform for elucidating structure–function relationships and mechanistic differences among members of this intriguing group of accessory subunits. Here, we specifically investigated the determinants of KCNQ1 inhibition by KCNE4, the least well-studied KCNE protein. In CHO-K1 cells, KCNQ1, but not KCNQ4, is strongly inhibited by coexpression with KCNE4. By studying KCNQ1-KCNQ4 chimeras, we identified two adjacent residues (K326 and T327) within the extracellular end of the KCNQ1 S6 segment that determine inhibition of KCNQ1 by KCNE4. This dipeptide motif is distinct from neighboring S6 sequences that enable modulation by KCNE1 and KCNE3. Conversely, S6 mutations (S338C and F340C) that alter KCNE1 and KCNE3 effects on KCNQ1 do not abrogate KCNE4 inhibition. Further, KCNQ1-KCNQ4 chimeras that exhibited resistance to the inhibitory effects of KCNE4 still interact biochemically with this protein, implying that accessory subunit binding alone is not sufficient for channel modulation. These observations indicate that the diverse functional effects observed for KCNE proteins depend, in part, on structures intrinsic to the pore-forming subunit, and that distinct S6 subdomains determine KCNQ1 responses to KCNE1, KCNE3, and KCNE4.

Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv

Mon, 31 Aug 2009 12:46:13 PDT

Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K⁺ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K⁺ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K⁺, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. "Fast" gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a "slow" gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current.

Different pathways for activation and deactivation in CaV1.2: a minimal gating model

Beyl, S., Kugler, P., Kudrnac, M., Hohaus, A., Hering, S., Timin, E. — Mon, 31 Aug 2009 12:46:13 PDT

Point mutations in pore-lining S6 segments of Ca_V1.2 shift the voltage dependence of activation into the hyperpolarizing direction and significantly decelerate current activation and deactivation. Here, we analyze theses changes in channel gating in terms of a circular four-state model accounting for an activation R–A–O and a deactivation O–D–R pathway. Transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements. Voltage-independent pore openings and closures during activation (A–O) and deactivation (D–R) are described by rate constants and β, and and , respectively. Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)). Estimated model parameters of 15 Ca_V1.2 constructs well describe the activation and deactivation processes. Voltage dependence of the "pore-releasing" sensor movement ((x(V)) was much weaker than the voltage dependence of "pore-locking" sensor movement (y(V)). Our data suggest that changes in membrane voltage are more efficient in closing than in opening Ca_V1.2. The model failed to reproduce current kinetics of mutation A780P that was, however, accurately fitted with individually adjusted x(V) and y(V). We speculate that structural changes induced by a proline substitution in this position may disturb the voltage-sensing domain.

Proton inhibition of unitary currents of vanilloid receptors

Liu, B., Yao, J., Wang, Y., Li, H., Qin, F. — Mon, 31 Aug 2009 12:46:13 PDT

Protons, which are released during inflammation and injury, regulate many receptors and ion channels involved in pain transduction, including capsaicin channels (transient receptor potential vanilloid receptors 1). Whereas extracellular acidification both sensitizes and directly activates the channel, it also causes concomitant reduction of the unitary current amplitudes. Here, we investigate the mechanisms and molecular basis of this inhibitory effect of protons on channel conductance. Single-channel recordings showed that the unitary current amplitudes decreased with extracellular pH in a dose-dependent manner, consistent with a model in which protons bind to a site within the channel with an apparent pKa of ~6. The inhibition was voltage dependent, ~65% at –60 mV and 37% at +60 mV when pH was reduced from 7.4 to 5.5. The unitary current amplitudes reached saturation at [K⁺] ≥ 1 M, and notably the maximum amplitudes did not converge with different pHs, inconsistent with a blockade model based on surface charge screening or competitive inhibition of permeating ions. Mutagenesis experiments uncovered two acidic residues critical for proton inhibition, one located at the pore entrance and the other on the pore helix. Based on homology to the KcsA structure, the two acidic residues, along with another basic residue also on the pore helix, could form a triad interacting with each other through extensive hydrogen bonds and electrostatic contacts, suggesting that protons may mediate the interactions between the selectivity filter and pore helix, thereby altering the local structure in the filter region and consequently the conductance of the channel.

Cooperative regulation of Cav1.2 channels by intracellular Mg2+, the proximal C-terminal EF-hand, and the distal C-terminal domain

Brunet, S., Scheuer, T., Catterall, W. A. — Mon, 27 Jul 2009 10:01:40 PDT

L-type Ca²⁺ currents conducted by Ca_v1.2 channels initiate excitation–contraction coupling in cardiac myocytes. Intracellular Mg²⁺ (Mg_i) inhibits the ionic current of Ca_v1.2 channels. Because Mg_i is altered in ischemia and heart failure, its regulation of Ca_v1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mg_i on voltage-dependent inactivation (VDI) of Ca_v1.2 channels using Na⁺ as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca²⁺-dependent inactivation process. We confirmed that increased Mg_i reduces peak ionic currents and increases VDI of Ca_v1.2 channels in ventricular myocytes and in transfected cells when measured with Na⁺ as permeant ion. The increased rate and extent of VDI caused by increased Mg_i were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mg_i to the EF-hand (K_D 0.9 mM) near the resting level of Mg_i in ventricular myocytes. VDI was more rapid for L-type Ca²⁺ currents in ventricular myocytes than for Ca_v1.2 channels in transfected cells. Coexpression of Ca_vβ_2b subunits and formation of an autoinhibitory complex of truncated Ca_v1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Ca_v1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg_i binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca_v1.2 channels by Mg_i, the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.

The external pore loop interacts with S6 and S3-S4 linker in domain 4 to assume an essential role in gating control and anticonvulsant action in the Na+ channel

Yang, Y.-C., Hsieh, J.-Y., Kuo, C.-C. — Mon, 27 Jul 2009 10:01:40 PDT

Carbamazepine, phenytoin, and lamotrigine are widely prescribed anticonvulsants in neurological clinics. These drugs bind to the same receptor site, probably with the diphenyl motif in their structure, to inhibit the Na⁺ channel. However, the location of the drug receptor remains controversial. In this study, we demonstrate close proximity and potential interaction between an external aromatic residue (W1716 in the external pore loop) and an internal aromatic residue (F1764 in the pore-lining part of the sixth transmembrane segment, S6) of domain 4 (D4), both being closely related to anticonvulsant and/or local anesthetic binding to the Na⁺ channel. Double-mutant cycle analysis reveals significant cooperativity between the two phenyl residues for anticonvulsant binding. Concomitant F1764C mutation evidently decreases the susceptibility of W1716C to external Cd²⁺ and membrane-impermeable methanethiosulfonate reagents. Also, the W1716E/F1764R and G1715E/F1764R double mutations significantly alter the selectivity for Na⁺ over K⁺ and markedly shift the activation curve, respectively. W1716 and F1764 therefore very likely form a link connecting the outer and inner compartments of the Na⁺ channel pore (in addition to the selectivity filter). Anticonvulsants and local anesthetics may well traverse this "S6 recess" without trespassing on the selectivity filter. Furthermore, we found that Y1618K, a point mutation in the S3-4 linker (the extracellular extension of D4S4), significantly alters the consequences of carbamazepine binding to the Na⁺ channel. The effect of Y1618K mutation, however, is abolished by concomitant point mutations in the vicinity of Y1618, but not by those in the internally located inactivation machinery, supporting a direct local rather than a long-range allosteric action. Moreover, Y1618 could interact with D4 pore residues W1716 and L1719 to have a profound effect on both channel gating and anticonvulsant action. We conclude that there are direct interactions among the external S3-4 linker, the external pore loop, and the internal S6 segment in D4, making the external pore loop a pivotal point critically coordinating ion permeation, gating, and anticonvulsant binding in the Na⁺ channel.

The principal neurons of the medial nucleus of the trapezoid body and NG2+ glial cells receive coordinated excitatory synaptic input

Mon, 27 Jul 2009 10:01:40 PDT

Glial cell processes are part of the synaptic structure and sense spillover of transmitter, while some glial cells can even receive direct synaptic input. Here, we report that a defined type of glial cell in the medial nucleus of the trapezoid body (MNTB) receives excitatory glutamatergic synaptic input from the calyx of Held (CoH). This giant glutamatergic terminal forms an axosomatic synapse with a single principal neuron located in the MNTB. The NG2 glia, as postsynaptic principal neurons, establish synapse-like structures with the CoH terminal. In contrast to the principal neurons, which are known to receive excitatory as well as inhibitory inputs, the NG2 glia receive mostly, if not exclusively, -amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor–mediated evoked and spontaneous synaptic input. Simultaneous recordings from neurons and NG2 glia indicate that they partially receive synchronized spontaneous input. This shows that an NG2⁺ glial cell and a postsynaptic neuron share presynaptic terminals.

Application of rate-equilibrium free energy relationship analysis to nonequilibrium ion channel gating mechanisms

Csanady, L. — Mon, 27 Jul 2009 10:01:40 PDT

Rate-equilibrium free energy relationship (REFER) analysis provides information on transition-state structures and has been applied to reveal the temporal sequence in which the different regions of an ion channel protein move during a closed–open conformational transition. To date, the theory used to interpret REFER relationships has been developed only for equilibrium mechanisms. Gating of most ion channels is an equilibrium process, but recently several ion channels have been identified to have retained nonequilibrium traits in their gating cycles, inherited from transporter-like ancestors. So far it has not been examined to what extent REFER analysis is applicable to such systems. By deriving the REFER relationships for a simple nonequilibrium mechanism, this paper addresses whether an equilibrium mechanism can be distinguished from a nonequilibrium one by the characteristics of their REFER plots, and whether information on the transition-state structures can be obtained from REFER plots for gating mechanisms that are known to be nonequilibrium cycles. The results show that REFER plots do not carry information on the equilibrium nature of the underlying gating mechanism. Both equilibrium and nonequilibrium mechanisms can result in linear or nonlinear REFER plots, and complementarity of REFER slopes for opening and closing transitions is a trivial feature true for any mechanism. Additionally, REFER analysis provides limited information about the transition-state structures for gating schemes that are known to be nonequilibrium cycles.

The 9-methyl group of retinal is essential for rapid Meta II decay and phototransduction quenching in red cones

Mon, 27 Jul 2009 10:01:41 PDT

Cone photoreceptors of the vertebrate retina terminate their response to light much faster than rod photoreceptors. However, the molecular mechanisms underlying this rapid response termination in cones are poorly understood. The experiments presented here tested two related hypotheses: first, that the rapid decay rate of metarhodopsin (Meta) II in red-sensitive cones depends on interactions between the 9-methyl group of retinal and the opsin part of the pigment molecule, and second, that rapid Meta II decay is critical for rapid recovery from saturation of red-sensitive cones after exposure to bright light. Microspectrophotometric measurements of pigment photolysis, microfluorometric measurements of retinol production, and single-cell electrophysiological recordings of flash responses of salamander cones were performed to test these hypotheses. In all cases, cones were bleached and their visual pigment was regenerated with either 11-cis retinal or with 11-cis 9-demethyl retinal, an analogue of retinal lacking the 9-methyl group. Meta II decay was four to five times slower and subsequent retinol production was three to four times slower in red-sensitive cones lacking the 9-methyl group of retinal. This was accompanied by a significant slowing of the recovery from saturation in cones lacking the 9-methyl group after exposure to bright (>0.1% visual pigment photoactivated) but not dim light. A mathematical model of the turn-off process of phototransduction revealed that the slower recovery of photoresponse can be explained by slower Meta decay of 9-demethyl visual pigment. These results demonstrate that the 9-methyl group of retinal is required for steric chromophore–opsin interactions that favor both the rapid decay of Meta II and the rapid response recovery after exposure to bright light in red-sensitive cones.

Mutations reveal voltage gating of CNGA1 channels in saturating cGMP

Martinez-Francois, J. R., Xu, Y., Lu, Z. — Mon, 27 Jul 2009 10:01:41 PDT

Activity of cyclic nucleotide–gated (CNG) cation channels underlies signal transduction in vertebrate visual receptors. These highly specialized receptor channels open when they bind cyclic GMP (cGMP). Here, we find that certain mutations restricted to the region around the ion selectivity filter render the channels essentially fully voltage gated, in such a manner that the channels remain mostly closed at physiological voltages, even in the presence of saturating concentrations of cGMP. This voltage-dependent gating resembles the selectivity filter-based mechanism seen in KcsA K⁺ channels, not the S4-based mechanism of voltage-gated K⁺ channels. Mutations that render CNG channels gated by voltage loosen the attachment of the selectivity filter to its surrounding structure, thereby shifting the channel's gating equilibrium toward closed conformations. Significant pore opening in mutant channels occurs only when positive voltages drive the pore from a low-probability open conformation toward a second open conformation to increase the channels' open probability. Thus, the structure surrounding the selectivity filter has evolved to (nearly completely) suppress the expression of inherent voltage-dependent gating of CNGA1, ensuring that the binding of cGMP by itself suffices to open the channels at physiological voltages.