Toxin binding reveals two open state structures for one acid-sensing ion channel

Of the three principal conformations of acid-sensing ion channels (ASICs)—closed, open and desensitized—only the atomic structure of the desensitized conformation had been known. Two recent papers report the crystal structure of chicken ASIC1 in complex with the spider toxin psalmotoxin 1, and one of these studies finds that, depending on the pH, channels are in two different open conformations. Compared with the desensitized conformation, toxin binding induces only subtle structural changes in the lower part of the large extracellular domain but a complete rearrangement of the two transmembrane domains (TMDs), suggesting that desensitization gating (the transition from open to desensitized) is mainly associated with conformational rearrangements of the TMDs. Moreover, the study reveals how two different arrangements of the TMDs in the open state give rise to ion pores with different selectivity for monovalent cations.     

Candidate amino acids involved in H+ gating of acid-sensing ion channel 1a

Acid-sensing ion channels are ligand-gated cation channels, gated by extracellular H(+). H(+) is the simplest ligand possible, and whereas for larger ligands that gate ion channels complex binding sites in the three-dimensional structure of the proteins have to be assumed, H(+) could in principle gate a channel by titration of a single amino acid. Experimental evidence suggests a more complex situation, however. For example, it has been shown that extracellular Ca(2+) ions compete with H(+); probably Ca(2+) ions bound to the extracellular loop of ASICs stabilize the closed state of the channel and have to be displaced before the channel can open. In such a scheme, amino acids contributing to Ca(2+) binding would also be candidates contributing to H(+) gating. In this study we systematically screened more than 40 conserved, charged amino acids in the extracellular region of ASIC1a for a possible contribution to H(+) gating. We identified four amino acids where substitution strongly affects H(+) gating: Glu(63), His(72)/His(73), and Asp(78). These amino acids are highly conserved among H(+)-sensitive ASICs and are candidates for the "H(+) sensor" of ASICs.     

Acid sensing ion channels regulate neuronal excitability by inhibiting BK potassium channels

Acid sensing ion channels (ASICs), Ca²⁺ and voltage-activated potassium channels (BK) are widely present throughout the central nervous system. Previous studies have shown that when expressed together in heterologous cells, ASICs inhibit BK channels, and this inhibition is relieved by acidic extracellular pH. We hypothesized that ASIC and BK channels might interact in neurons, and that ASICs may regulate BK channel activity. We found that ASICs inhibited BK currents in cultured wild-type cortical neurons, but not in ASIC1a/2/3 triple knockout neurons. The inhibition in the wild-type was partially relieved by a drop in extracellular pH to 6. To test the consequences of ASIC-BK interaction for neuronal excitability, we compared action potential firing in cultured cortical neurons from wild-type and ASIC1a/2/3 null mice. We found that in the knockout, action potentials were narrow and exhibited increased after-hyperpolarization. Moreover, the excitability of these neurons was significantly increased. These findings are consistent with increased BK channel activity in the neurons from ASIC1a/2/3 null mice. Our data suggest that ASICs can act as endogenous pH-dependent inhibitors of BK channels, and thereby can reduce neuronal excitability.     

Calcium-permeable acid-sensing ion channel is a molecular target of the neurotoxic metal ion lead

Acid-sensing ion channels (ASICs) are emerging as fundamental players in the regulation of neural plasticity and in pathological conditions. Here we showed that lead (Pb2+), a well known neurotoxic metal ion, reversibly and concentration-dependently inhibited ASIC currents in the acutely dissociated spinal dorsal horn and hippocampal CA1 neurons of rats. In vitro expression of ASIC subunits in combination demonstrated that both ASIC1 and -3 subunits were sensitive to Pb2+. Mechanistically, Pb2+ reduced the pH sensitivity of ASICs independent of membrane voltage change. Moreover, Pb2+ inhibited the ASIC-mediated membrane depolarization and the elevation of intracellular Ca2+ concentration. In addition, we compared the effect of Pb2+ with that of Ca2+ or amiloride to explore the possible interactions of Pb2+ and Ca2+ in regulating ASICs, and we found that Pb2+ inhibited ASIC currents independent of the amiloride/Ca2+ blockade. Because ASIC1b and -3 subunits are mainly expressed in peripheral neurons, our data identified ASIC1a-containing Ca2+-permeable ASIC as a novel central target of Pb2+ action, which may contribute to Pb2+ neurotoxicity.     

Interaction of acid-sensing ion channel (ASIC) 1 with the tarantula toxin psalmotoxin 1 is state dependent

Acid-sensing ion channels (ASICs) are Na(+) channels gated by extracellular H(+). Six ASIC subunits that are expressed in neurons have been characterized. The tarantula toxin psalmotoxin 1 has been reported to potently and specifically inhibit homomeric ASIC1a and has been useful to characterize ASICs in neurons. Recently we have shown that psalmotoxin 1 inhibits ASIC1a by increasing its apparent affinity for H(+). However, the mechanism by which PcTx1 increases the apparent H(+) affinity remained unclear. Here we show that PcTx1 also interacts with ASIC1b, a splice variant of ASIC1a. However, PcTx1 does not inhibit ASIC1b but promotes its opening; under slightly acidic conditions, PcTx1 behaves like an agonist for ASIC1b. Our results are most easily explained by binding of PcTx1 with different affinities to different states (closed, open, and desensitized) of the channel. For ASIC1b, PcTx1 binds most tightly to the open state, promoting opening, whereas for ASIC1a, it binds most tightly to the open and the desensitized state, promoting desensitization.     

Toxin binding reveals two open state structures for one acid-sensing ion channel

Of the three principal conformations of acid-sensing ion channels (ASICs)—closed, open and desensitized—only the atomic structure of the desensitized conformation had been known. Two recent papers report the crystal structure of chicken ASIC1 in complex with the spider toxin psalmotoxin 1, and one of these studies finds that, depending on the pH, channels are in two different open conformations. Compared with the desensitized conformation, toxin binding induces only subtle structural changes in the lower part of the large extracellular domain but a complete rearrangement of the two transmembrane domains (TMDs), suggesting that desensitization gating (the transition from open to desensitized) is mainly associated with conformational rearrangements of the TMDs. Moreover, the study reveals how two different arrangements of the TMDs in the open state give rise to ion pores with different selectivity for monovalent cations.     

Gating of acid-sensitive ion channel-1: release of Ca2+ block vs. allosteric mechanism

The acid-sensitive ion channels (ASICs) are a family of voltage-insensitive sodium channels activated by external protons. A previous study proposed that the mechanism underlying activation of ASIC consists of the removal of a Ca2+ ion from the channel pore (Immke and McCleskey, 2003). In this work we have revisited this issue by examining single channel recordings of ASIC1 from toadfish (fASIC1). We demonstrate that increases in the concentration of external protons or decreases in the concentration of external Ca2+ activate fASIC1 by progressively opening more channels and by increasing the rate of channel opening. Both maneuvers produced similar effects in channel kinetics, consistent with the former notion that protons displace a Ca2+ ion from a high-affinity binding site. However, we did not observe any of the predictions expected from the release of an open-channel blocker: decrease in the amplitude of the unitary currents, shortening of the mean open time, or a constant delay for the first opening when the concentration of external Ca2+ was decreased. Together, the results favor changes in allosteric conformations rather than unblocking of the pore as the mechanism gating fASIC1. At high concentrations, Ca2+ has an additional effect that consists of voltage-dependent decrease in the amplitude of unitary currents (EC50 of 10 mM at -60 mV and pH 6.0). This phenomenon is consistent with voltage-dependent block of the pore but it occurs at concentrations much higher than those required for gating.     

Selective regulation of acid-sensing ion channel 1 by serine proteases

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that belong to the epithelial Na(+) channel/degenerin family. ASICs are transiently activated by a rapid drop in extracellular pH. Conditions of low extracellular pH, such as ischemia and inflammation in which ASICs are thought to be active, are accompanied by increased protease activity. We show here that serine proteases modulate the function of ASIC1a and ASIC1b but not of ASIC2a and ASIC3. We show that protease exposure shifts the pH dependence of ASIC1a activation and steady-state inactivation to more acidic pH. As a consequence, protease exposure leads to a decrease in current response if ASIC1a is activated by a pH drop from pH 7.4. If, however, acidification occurs from a basal pH of approximately 7, protease-exposed ASIC1a shows higher activity than untreated ASIC1a. We provide evidence that this bi-directional regulation of ASIC1a function also occurs in neurons. Thus, we have identified a mechanism that modulates ASIC function and may allow ASIC1a to adapt its gating to situations of persistent extracellular acidification.     

Zebrafish acid-sensing ion channel (ASIC) 4, characterization of homo- and heteromeric channels, and identification of regions important for activation by H+

There are four genes for acid-sensing ion channels (ASICs) in the genome of mammalian species. Whereas ASIC1 to ASIC3 form functional H+-gated Na+ channels, ASIC4 is not gated by H+, and its function is unknown. Zebrafish has two ASIC4 paralogs: zASIC4.1 and zASIC4.2. Whereas zASIC4.1 is gated by extracellular H+, zASIC4.2 is not. This differential response to H+ makes zASIC4 paralogs a good model to study the properties of this ion channel. In this study, we found that surface expression of homomeric zASIC4.2 is higher than that of zASIC4.1. Surface expression of zASIC4.1 was much increased by formation of heteromeric channels, suggesting that zASIC4.1 contributes to heteromeric ASICs in zebrafish neurons. Robust surface expression of H+-insensitive zASIC4.2 suggests that zASIC4.2 functions as a homomer and is gated by an as yet unknown stimulus, different from H+. Moreover, we identified a small region just distal to the first transmembrane domain that is crucial for the differential H+ response of the two paralogs. This post-TM1 domain may have a general role in gating of members of this gene family.     

Gating and conductance properties of BK channels are modulated by the S9-S10 tail domain of the alpha subunit. A study of mSlo1 and mSlo3 wild-type and chimeric channels.

The COOH-terminal S9-S10 tail domain of large conductance Ca(2+)-activated K(+) (BK) channels is a major determinant of Ca(2+) sensitivity (Schreiber, M., A. Wei, A. Yuan, J. Gaut, M. Saito, and L. Salkoff. 1999. Nat. Neurosci. 2:416-421). To investigate whether the tail domain also modulates Ca(2+)-independent properties of BK channels, we explored the functional differences between the BK channel mSlo1 and another member of the Slo family, mSlo3 (Schreiber, M., A. Yuan, and L. Salkoff. 1998. J. Biol. Chem. 273:3509-3516). Compared with mSlo1 channels, mSlo3 channels showed little Ca(2+) sensitivity, and the mean open time, burst duration, gaps between bursts, and single-channel conductance of mSlo3 channels were only 32, 22, 41, and 37% of that for mSlo1 channels, respectively. To examine which channel properties arise from the tail domain, we coexpressed the core of mSlo1 with either the tail domain of mSlo1 or the tail domain of mSlo3 channels, and studied the single-channel currents. Replacing the mSlo1 tail with the mSlo3 tail resulted in the following: increased open probability in the absence of Ca(2+); reduced the Ca(2+) sensitivity greatly by allowing only partial activation by Ca(2+) and by reducing the Hill coefficient for Ca(2+) activation; decreased the voltage dependence approximately 28%; decreased the mean open time two- to threefold; decreased the mean burst duration three- to ninefold; decreased the single-channel conductance approximately 14%; decreased the K(d) for block by TEA(i) approximately 30%; did not change the minimal numbers of three to four open and five to seven closed states entered during gating; and did not change the major features of the dependency between adjacent interval durations. These observations support a modular construction of the BK channel in which the tail domain modulates the gating kinetics and conductance properties of the voltage-dependent core domain, in addition to determining most of the high affinity Ca(2+) sensitivity.     

Human ether-à-go-go-related gene K+ channel gating probed with extracellular ca2+. Evidence for two distinct voltage sensors

Human ether-à-go-go-related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 +/- 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-à-go-go-related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery.     

Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that are members of the epithelial Na(+) channel/degenerin family and are transiently activated by extracellular acidification. ASICs in the central nervous system have a modulatory role in synaptic transmission and are involved in cell injury induced by acidosis. We have recently demonstrated that ASIC function is regulated by serine proteases. We provide here evidence that this regulation of ASIC function is tightly linked to channel cleavage. Trypsin cleaves ASIC1a with a similar time course as it changes ASIC1a function, whereas ASIC1b, whose function is not modified by trypsin, is not cleaved. Trypsin cleaves ASIC1a at Arg-145, in the N-terminal part of the extracellular loop, between a highly conserved sequence and a sequence that is critical for ASIC1a inhibition by the venom of the tarantula Psalmopoeus cambridgei. This channel domain controls the inactivation kinetics and co-determines the pH dependence of ASIC gating. It undergoes a conformational change during inactivation, which renders the cleavage site inaccessible to trypsin in inactivated channels.     

Modal gating of human CaV2.1 (P/Q-type) calcium channels: II. the b mode and reversible uncoupling of inactivation

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing these calcium channels. Human CaV2.1 channels showed a complex modal gating, which is described in this and the preceding paper (Luvisetto, S., T. Fellin, M. Spagnolo, B. Hivert, P.F. Brust, M.M. Harpold, K.A. Stauderman, M.E. Williams, and D. Pietrobon. 2004. J. Gen. Physiol. 124:445-461). Here, we report the characterization of the so-called b gating mode. A CaV2.1 channel in the b gating mode shows a bell-shaped voltage dependence of the open probability, and a characteristic low open probability at high positive voltages, that decreases with increasing voltage, as a consequence of both shorter mean open time and longer mean closed time. Reversible transitions of single human CaV2.1 channels between the b gating mode and the mode of gating in which the channel shows the usual voltage dependence of the open probability (nb gating mode) were much more frequent (time scale of seconds) than those between the slow and fast gating modes (time scale of minutes; Luvisetto et al., 2004), and occurred independently of whether the channel was in the fast or slow mode. We show that the b gating mode produces reversible uncoupling of inactivation in human CaV2.1 channels. In fact, a CaV2.1 channel in the b gating mode does not inactivate during long pulses at high positive voltages, where the same channel in both fast-nb and slow-nb gating modes inactivates relatively rapidly. Moreover, a CaV2.1 channel in the b gating mode shows a larger availability to open than in the nb gating modes. Regulation of the complex modal gating of human CaV2.1 channels could be a potent and versatile mechanism for the modulation of synaptic strength and plasticity as well as of neuronal excitability and other postsynaptic Ca2+-dependent processes.     

Spontaneous channel activity of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Application of allosteric modeling to calcium and InsP3 regulation of InsP3R single-channel gating

The InsP3R Ca2+ release channel has a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca2+]i. To determine if relieving Ca2+ inhibition is sufficient for channel activation, we examined single-channel activities in low [Ca2+]i in the absence of InsP3, by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent channel activities with low open probability Po ( approximately 0.03) were observed in [Ca2+]i < 5 nM with the same frequency as in the presence of InsP3, whereas no activities were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i of the channel to be 1.2-4.0 nM in the absence of InsP3, and demonstrate that the channel can be active when all of its ligand-binding sites (including InsP3) are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies of [Ca2+]i and InsP3 regulation of steady-state channel gating behavior of types 1 and 3 InsP3R isoforms, including spontaneous InsP3-independent channel activities, the tetrameric channel can adopt six different conformations, the equilibria among which are controlled by two inhibitory and one activating Ca2+-binding and one InsP3-binding sites in a manner outlined in the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the Ca2+ affinities of the high-affinity inhibitory sites in different conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent low-affinity inhibitory site. The model also suggests that besides the ligand-regulated gating mechanism, the channel has a ligand-independent gating mechanism responsible for maximum channel Po being less than unity. The validity of this model was established by its successful quantitative prediction of channel behavior after it had been exposed to ultra-low bath [Ca2+].     

Protons open acid-sensing ion channels by catalyzing relief of Ca2+ blockade

Acid-sensing ion channels (ASICs) open when extracellular pH drops and they are enhanced by lactate, making them specialized for detecting lactic acidosis. Highly expressed on cardiac nociceptors and some other sensory neurons, ASICs may help trigger pain caused by tissue ischemia. We report that H(+) opens ASIC3 by speeding release of Ca(2+) from a high-affinity binding site (K(Ca) = 150 nM) on the extracellular side of the pore. The bound Ca(2+) blocks permeation and the channel conducts when multiple H(+) ions relieve this block. Activation through Ca(2+) explains sensitivity to lactate, which decreases extracellular [Ca(2+)], and it may prove relevant in CNS pathologies (stroke, seizure) that simultaneously drop pH and Ca(2+).     

Role of the beta1 subunit in large-conductance Ca(2+)-activated K(+) channel gating energetics. Mechanisms of enhanced Ca(2+) sensitivity

Over the past few years, it has become clear that an important mechanism by which large-conductance Ca²⁺-activated K⁺ channel (BK_Ca) activity is regulated is the tissue-specific expression of auxiliary β subunits. The first of these to be identified, β1, is expressed predominately in smooth muscle and causes dramatic effects, increasing the apparent affinity of the channel for Ca²⁺ 10-fold at 0 mV, and shifting the range of voltages over which the channel activates −80 mV at 9.1 μM Ca²⁺. With this study, we address the question: which aspects of BK_Ca gating are altered by β1 to bring about these effects: Ca²⁺ binding, voltage sensing, or the intrinsic energetics of channel opening? The approach we have taken is to express the β1 subunit together with the BK_Ca α subunit in Xenopus oocytes, and then to compare β1''s steady state effects over a wide range of Ca²⁺ concentrations and membrane voltages to those predicted by allosteric models whose parameters have been altered to mimic changes in the aspects of gating listed above. The results of our analysis suggest that much of β1''s steady state effects can be accounted for by a reduction in the intrinsic energy the channel must overcome to open and a decrease in its voltage sensitivity, with little change in the affinity of the channel for Ca²⁺ when it is either open or closed. Interestingly, however, the small changes in Ca²⁺ binding affinity suggested by our analysis (K_c 7.4 μM → 9.6 μM; K_o = 0.80 μM → 0.65 μM) do appear to be functionally important. We also show that β1 affects the mSlo conductance–voltage relation in the essential absence of Ca²⁺, shifting it +20 mV and reducing its apparent gating charge 38%, and we develop methods for distinguishing between alterations in Ca²⁺ binding and other aspects of BK_Ca channel gating that may be of general use.     

Single channel properties of rat acid-sensitive ion channel-1alpha, -2a,and -3 expressed in Xenopus oocytes

The mammalian nervous system expresses proton-gated ion channels known as acid-sensing ion channels (ASICs). Depending on their location and specialization some neurons express more than one type of ASIC where they may form homo- or heteromeric channels. Macroscopic characteristics of the ASIC currents have been described, but little is known at the single channel level. Here, we have examined the properties of unitary currents of homomeric rat ASIC1alpha, ASIC2a, and ASIC3 expressed in Xenopus oocytes with the patch clamp technique. We describe and characterize properties unique to each of these channels that can be used to distinguish the various types of ASIC channels expressed in mammalian neurons. The amplitudes of the unitary currents in symmetrical Na(+) are similar for the three types of channels (23-18 pS) and are not voltage dependent. However, ASIC1alpha exhibits three subconductance states, ASIC2a exhibits only one, and ASIC3 none. The kinetics of the three types of channels are different: ASIC1alpha and ASIC2a shift between modes of activity, each mode has different open probability and kinetics. In contrast, the kinetics of ASIC3 are uniform throughout the burst of activity. ASIC1alpha, ASIC2a, and ASIC3 are activated by external protons with apparent pH(50) of 5.9, 5.0, and 5.4, respectively. Desensitization in the continual presence of protons is fast and complete in ASIC1alpha and ASIC3 (2.0 and 4.5 s(-1), respectively) but slow and only partial in ASIC2a (0.045 s(-1)). The response to external Ca(2+) also differs: micro M concentrations of extracellular Ca(2+) are necessary for proton gating of ASIC3 (EC(50) = 0.28 micro M), whereas ASIC1alpha and ASIC2a do not require Ca(2+). In addition, Ca(2+) inhibits ASIC1alpha (K(D) = 9.2 +/- 2 mM) by several mechanisms: decrease in the amplitude of unitary currents, shortening of the burst of activity, and decrease in the number of activated channels. Contrary to previous reports, our results indicate that the Ca(2+) permeability of ASIC1alpha is very small.     

A store-operated calcium channel in Drosophila S2 cells

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca(2+)-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca(2+)-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20-50 pA at -110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing approximately 300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ > Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of approximately 50 nM, and was also blocked by 20 microM SKF 96365 and by 20 microM 2-APB. At concentrations between 5 and 14 microM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.     

ATP-dependent adenophostin activation of inositol 1,4,5-trisphosphate receptor channel gating: kinetic implications for the durations of calcium puffs in cells

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) is a ligand-gated intracellular Ca(2+) release channel that plays a central role in modulating cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)). The fungal metabolite adenophostin A (AdA) is a potent agonist of the InsP(3)R that is structurally different from InsP(3) and elicits distinct calcium signals in cells. We have investigated the effects of AdA and its analogues on single-channel activities of the InsP(3)R in the outer membrane of isolated Xenopus laevis oocyte nuclei. InsP(3)R activated by either AdA or InsP(3) have identical channel conductance properties. Furthermore, AdA, like InsP(3), activates the channel by tuning Ca(2+) inhibition of gating. However, gating of the AdA-liganded InsP(3)R has a critical dependence on cytoplasmic ATP free acid concentration not observed for InsP(3)-liganded channels. Channel gating activated by AdA is indistinguishable from that elicited by InsP(3) in the presence of 0.5 mM ATP, although the functional affinity of the channel is 60-fold higher for AdA. However, in the absence of ATP, gating kinetics of AdA-liganded InsP(3)R were very different. Channel open time was reduced by 50%, resulting in substantially lower maximum open probability than channels activated by AdA in the presence of ATP, or by InsP(3) in the presence or absence of ATP. Also, the higher functional affinity of InsP(3)R for AdA than for InsP(3) is nearly abolished in the absence of ATP. Low affinity AdA analogues furanophostin and ribophostin activated InsP(3)R channels with gating properties similar to those of AdA. These results provide novel insights for interpretations of observed effects of AdA on calcium signaling, including the mechanisms that determine the durations of elementary Ca(2+) release events in cells. Comparisons of single-channel gating kinetics of the InsP(3)R activated by InsP(3), AdA, and its analogues also identify molecular elements in InsP(3)R ligands that contribute to binding and activation of channel gating.     

Interaction of the BKCa channel gating ring with dendrotoxins

Two classes of small homologous basic proteins, mamba snake dendrotoxins (DTX) and bovine pancreatic trypsin inhibitor (BPTI), block the large conductance Ca²⁺-activated K⁺ channel (BK_Ca, KCa1.1) by production of discrete subconductance events when added to the intracellular side of the membrane. This toxin-channel interaction is unlikely to be pharmacologically relevant to the action of mamba venom, but as a fortuitous ligand-protein interaction, it has certain biophysical implications for the mechanism of BK_Ca channel gating. In this work we examined the subconductance behavior of 9 natural dendrotoxin homologs and 6 charge neutralization mutants of δ-dendrotoxin in the context of current structural information on the intracellular gating ring domain of the BK_Ca channel. Calculation of an electrostatic surface map of the BK_Ca gating ring based on the Poisson-Boltzmann equation reveals a predominantly electronegative surface due to an abundance of solvent-accessible side chains of negatively charged amino acids. Available structure-activity information suggests that cationic DTX/BPTI molecules bind by electrostatic attraction to site(s) on the gating ring located in or near the cytoplasmic side portals where the inactivation ball peptide of the β2 subunit enters to block the channel. Such an interaction may decrease the apparent unitary conductance by altering the dynamic balance of open versus closed states of BK_Ca channel activation gating.