The effect of wilderness and medical training on injury and altitude preparedness among backcountry hikers in Rocky Mountain National Park / 世界急诊医学杂志（英文）

BACKGROUND:The purpose of this study was to document the correlation between medical and wilderness training with levels of preparedness for acute mountain sickness (AMS), illness, and injury among backcountry hikers. METHODS:We conducted a cross-sectional, convenience survey in Rocky Mountain National Park in July and August 2015. The study group consisted of 380 hikers who completed a written survey that collected information about demographics, wilderness experience, altitude experience, hiking equipment, communications devices, and trip planning. RESULTS:Factors such as wilderness training (wilderness first aid [WFA], wilderness first responder [WFR], or wilderness emergency medical technician [WEMT]), wilderness experience, and altitude experience all affected hikers' emergency preparedness. Respondents with medical training were more prepared to avoid or respond to AMS (62.3％ vs. 34.3％ [P<0.001]). They were also more prepared to avoid or manage injury/illness than hikers without medical training (37.7％vs. 20.7％ [P=0.003]). Participants with wilderness training were more likely to be prepared to avoid or respond to AMS (52.3％ vs. 36.8％ [P=0.025]) but not significantly more likely to be prepared to manage illness/injury (31.8％ vs. 22.0％ [P<0.11]). Adjusting for experience, wilderness training, age, and gender, we found that medical training was associated with increased preparedness for AMS (OR 2.72; 95％ CI 1.51–4.91) and injury/illness (OR 2.71; 95％ CI 1.5–4.89). CONCLUSION:Medically trained hikers were more likely to be prepared to avoid or manage AMS, medical emergencies, and injuries than their non-medically trained counterparts. Wilderness training increased hikers' preparedness for AMS but did not significantly alter preparedness for illness/injury.

Robust approaches to quantitative ratiometric FRET imaging of CFP/YFP fluorophores under confocal microscopy.

Ratiometric quantification of CFP/YFP FRET enables live-cell time-series detection of molecular interactions, without the need for acceptor photobleaching or specialized equipment for determining fluorescence lifetime. Although popular in widefield applications, its implementation on a confocal microscope, which would enable sub-cellular resolution, has met with limited success. Here, we characterize sources of optical variability (unique to the confocal context) that diminish the accuracy and reproducibility of ratiometric FRET determination and devise practical remedies. Remarkably, we find that the most popular configuration, which pairs an oil objective with a small pinhole aperture, results in intractable variability that could not be adequately corrected through any calibration procedure. By quantitatively comparing several imaging configurations and calibration procedures, we find that significant improvements can be achieved by combining a water objective and increased pinhole aperture with a uniform-dye calibration procedure. The combination of these methods permitted remarkably consistent quantification of sub-cellular FRET in live cells. Notably, this methodology can be readily implemented on a standard confocal instrument, and the dye calibration procedure yields a time savings over traditional live-cell calibration methods. In all, identification of key technical challenges and practical compensating solutions promise robust sub-cellular ratiometric FRET imaging under confocal microscopy.

Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells.

Among the most intriguing forms of Ca(2+) channel modulation is the regulation of L-type and P/Q-type channels by intracellular Ca(2+), acting via unconventional channel-calmodulin (CaM) interactions. In particular, overexpressing Ca(2+)-insensitive mutant CaM abolishes Ca(2+)-dependent modulation, hinting that Ca(2+)-free CaM may "preassociate" with these channels to enhance detection of local Ca(2+). Despite the far-reaching consequences of this proposal, in vitro experiments testing for preassociation provide conflicting results. Here, we develop a three filter-cube fluorescence resonance energy transfer method (three-cube FRET) to directly probe for constitutive associations between channel subunits and CaM in single living cells. This FRET assay detects Ca(2+)-independent associations between CaM and the pore-forming alpha(1) subunit of L-type, P/Q-type, and, surprisingly, R-type channels. These results now definitively demonstrate channel-CaM preassociation in resting cells and underscore the potential of three-cube FRET for probing protein-protein interactions.

Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels.

Acute modulation of P/Q-type (alpha1A) calcium channels by neuronal activity-dependent changes in intracellular Ca2+ concentration may contribute to short-term synaptic plasticity, potentially enriching the neurocomputational capabilities of the brain. An unconventional mechanism for such channel modulation has been proposed in which calmodulin (CaM) may exert two opposing effects on individual channels, initially promoting ('facilitation') and then inhibiting ('inactivation') channel opening. Here we report that such dual regulation arises from surprising Ca2+-transduction capabilities of CaM. First, although facilitation and inactivation are two competing processes, both require Ca2+-CaM binding to a single 'IQ-like' domain on the carboxy tail of alpha1A; a previously identified 'CBD' CaM-binding site has no detectable role. Second, expression of a CaM mutant with impairment of all four of its Ca2+-binding sites (CaM1234) eliminates both forms of modulation. This result confirms that CaM is the Ca2+ sensor for channel regulation, and indicates that CaM may associate with the channel even before local Ca2+ concentration rises. Finally, the bifunctional capability of CaM arises from bifurcation of Ca2+ signalling by the lobes of CaM: Ca2+ binding to the amino-terminal lobe selectively initiates channel inactivation, whereas Ca2+ sensing by the carboxy-terminal lobe induces facilitation. Such lobe-specific detection provides a compact means to decode local Ca2+ signals in two ways, and to separately initiate distinct actions on a single molecular complex.

G-protein inhibition of N- and P/Q-type calcium channels: distinctive elementary mechanisms and their functional impact.

Voltage-dependent G-protein inhibition of presynaptic Ca(2+) channels is a key mechanism for regulating synaptic efficacy. G-protein betagamma subunits produce such inhibition by binding to and shifting channel opening patterns from high to low open probability regimes, known respectively as "willing" and "reluctant" modes of gating. Recent macroscopic electrophysiological data hint that only N-type, but not P/Q-type channels can open in the reluctant mode, a distinction that could enrich the dimensions of synaptic modulation arising from channel inhibition. Here, using high-resolution single-channel recording of recombinant channels, we directly distinguished this core contrast in the prevalence of reluctant openings. Single, inhibited N-type channels manifested relatively infrequent openings of submillisecond duration (reluctant openings), which differed sharply from the high-frequency, millisecond gating events characteristic of uninhibited channels. By contrast, inhibited P/Q-type channels were electrically silent at the single-channel level. The functional impact of the differing inhibitory mechanisms was revealed in macroscopic Ca(2+) currents evoked with neuronal action potential waveforms (APWs). Fitting with a change in the manner of opening, inhibition of such N-type currents produced both decreased current amplitude and temporally advanced waveform, effects that would not only reduce synaptic efficacy, but also influence the timing of synaptic transmission. On the other hand, inhibition of P/Q-type currents evoked by APWs showed diminished amplitude without shape alteration, as expected from a simple reduction in the number of functional channels. Variable expression of N- and P/Q-type channels at spatially distinct synapses therefore offers the potential for custom regulation of both synaptic efficacy and synchrony, by G-protein inhibition.

Release-independent short-term synaptic depression in cultured hippocampal neurons.

Short-term synaptic plasticity may dramatically influence neuronal information transfer, yet the underlying mechanisms remain incompletely understood. In autapses (self-synapses) formed by cultured hippocampal neurons, short-term synaptic depression (STD) had several unusual features. (1) Reduction of neurotransmitter release probability with Cd(2+), a blocker of voltage-gated calcium channels, did not change depression. (2) Lowering [Ca(2+)](o) and/or raising [Mg(2+)](o) had little effect on STD in cells with strong baseline depression, but in cells with more modest baseline depression, it reduced the depression. (3) Random variations in the size of initial EPSCs did not influence successive EPSC sizes. These findings were inconsistent with release-dependent mechanisms, such as vesicle depletion, post-synaptic receptor desensitization, and autoreceptor inhibition. Instead, other results suggested that changes in action potentials (APs) contributed to depression. The somatic APs declined in amplitude with repetitive stimulation, and modest reduction of AP amplitudes with tetrodotoxin inhibited EPSCs. Notably, tetrodotoxin also increased depression. Similar changes in axonal APs could produce STD in at least two ways. First, decreasing presynaptic spike amplitudes could reduce calcium entry and release probability. Alternatively, APs could fail to propagate through some axonal branches, reducing the number of active synapses. To explore these possibilities, we derived the expected variance of EPSCs for the two scenarios. Experimentally, the variance increased and then decreased on average with successive responses during trains of APs, confirming a unique prediction from the conduction failure scenario. Thus, STD had surprising properties, incompatible with commonly postulated mechanisms but consistent with AP conduction failure at axonal branches.

Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels.

L-type (alpha(1C)) calcium channels inactivate rapidly in response to localized elevation of intracellular Ca(2+), providing negative Ca(2+) feedback in a diverse array of biological contexts. The dominant Ca(2+) sensor for such Ca(2+)-dependent inactivation has recently been identified as calmodulin, which appears to be constitutively tethered to the channel complex. This Ca(2+) sensor induces channel inactivation by Ca(2+)-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of alpha(1C). Apart from the IQ region, another crucial site for Ca(2+) inactivation appears to be a consensus Ca(2+)-binding, EF-hand motif, located approximately 100 amino acids upstream on the carboxyl terminus. However, the importance of this EF-hand motif for channel inactivation has become controversial since the original report from our lab implicating a critical role for this domain. Here, we demonstrate not only that the consensus EF hand is essential for Ca(2+) inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca(2+) inactivation. Mutating these amino acids to their counterparts in non-inactivating alpha(1E) calcium channels (MYEM) almost completely ablates Ca(2+) inactivation. In fact, only a single amino acid change of the second valine within this cluster to tyrosine (V1548Y) supports much of the functional knockout. However, mutations of presumed Ca(2+)-coordinating residues in the consensus EF hand reduce Ca(2+) inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca(2+) sensor, in which Ca(2+) binds according to a classic mechanism. We therefore suggest that while CaM serves as Ca(2+) sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of Ca(2+)-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca(2+)-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent Ca(2+) binding of CaM.

Ca(2+) channel modulation by recombinant auxiliary beta subunits expressed in young adult heart cells.

L-type Ca(2+) channels contribute importantly to the normal excitation-contraction coupling of physiological hearts, and to the functional derangement seen in heart failure. Although Ca(2+) channel auxiliary beta(1-4) subunits are among the strongest modulators of channel properties, little is known about their role in regulating channel behavior in actual heart cells. Current understanding draws almost exclusively from heterologous expression of recombinant subunits in model systems, which may differ from cardiocytes. To study beta-subunit effects in the cardiac setting, we here used an adenoviral-component gene-delivery strategy to express recombinant beta subunits in young adult ventricular myocytes cultured from 4- to 6-week-old rats. The main results were the following. (1) A component system of replication-deficient adenovirus, poly-L-lysine, and expression plasmids encoding beta subunits could be optimized to transfect young adult myocytes with 1% to 10% efficiency. (2) A reporter gene strategy based on green fluorescent protein (GFP) could be used to identify successfully transfected cells. Because fusion of GFP to beta subunits altered intrinsic beta-subunit properties, we favored the use of a bicistronic expression plasmid encoding both GFP and a beta subunit. (3) Despite the heteromultimeric composition of L-type channels (composed of alpha(1C), beta, and alpha(2)delta), expression of recombinant beta subunits alone enhanced Ca(2+) channel current density up to 3- to 4-fold, which argues that beta subunits are "rate limiting" for expression of current in heart. (4) Overexpression of the putative "cardiac" beta(2a) subunit more than halved the rate of voltage-dependent inactivation at +10 mV. This result demonstrates that beta subunits can tune inactivation in the myocardium and suggests that other beta subunits may be functionally dominant in the heart. Overall, this study points to the possible therapeutic potential of beta subunits to ameliorate contractile dysfunction and excitability in heart failure.

Differential occurrence of reluctant openings in G-protein-inhibited N- and P/Q-type calcium channels.

Voltage-dependent inhibition of N- and P/Q-type calcium channels by G proteins is crucial for presynaptic inhibition of neurotransmitter release, and may contribute importantly to short-term synaptic plasticity. Such calcium-channel modulation could thereby impact significantly the neuro-computational repertoire of neural networks. The differential modulation of N and P/Q channels could even further enrich their impact upon synaptic tuning. Here, we performed in-depth comparison of the G-protein inhibition of recombinant N and P/Q channels, expressed in HEK 293 cells with the m2 muscarinic receptor. While both channel types display classic features of G-protein modulation (kinetic slowing of activation, prepulse facilitation, and voltage dependence of inhibition), we confirmed previously reported quantitative differences, with N channels displaying stronger inhibition and greater relief of inhibition by prepulses. A more fundamental, qualitative difference in the modulation of these two channels was revealed by a modified tail-activation paradigm, as well as by a novel "slope" analysis method comparing time courses of slow activation and prepulse facilitation. The stark contrast in modulatory behavior can be understood within the context of the "willing-reluctant" model, in which binding of G-protein betagamma subunits to channels induces a reluctant mode of gating, where stronger depolarization is required for opening. Our experiments suggest that only N channels could be opened in the reluctant mode, at voltages normally spanned by neuronal action potentials. By contrast, P/Q channels appear to remain closed, especially over these physiological voltages. Further, the differential occurrence of reluctant openings is not explained by differences in the rate of G-protein unbinding from the two channels. These two scenarios predict very different effects of G-protein inhibition on the waveform of Ca(2+) entry during action potentials, with potentially important consequences for the timing and efficacy of synaptic transmission.

Relief of G-protein inhibition of calcium channels and short-term synaptic facilitation in cultured hippocampal neurons.

G-protein inhibition of voltage-gated calcium channels can be transiently relieved by repetitive physiological stimuli. Here, we provide evidence that such relief of inhibition contributes to short-term synaptic plasticity in microisland-cultured hippocampal neurons. With G-protein inhibition induced by the GABA(B) receptor agonist baclofen or the adenosine A1 receptor agonist 2-chloroadenosine, short-term synaptic facilitation emerged during action potential trains. The facilitation decayed with a time constant of approximately 100 msec. However, addition of the calcium channel inhibitor Cd(2+) at 2-3 microM had no such effect and did not alter baseline synaptic depression. As expected of facilitation from relief of channel inhibition, analysis of miniature EPSCs implicated presynaptic modulation, and elevating presynaptic Ca(2+) entry blunted the facilitation. Most telling was the near occlusion of synaptic facilitation after selective blockade of P/Q- but not N-type calcium channels. This was as predicted from experiments using recombinant calcium channels expressed in human embryonic kidney (HEK) 293 cells; we found significantly stronger relief of G-protein inhibition in recombinant P/Q- versus N-type channels during action potential trains. G-protein inhibition in HEK 293 cells was induced via recombinant M2 muscarinic acetylcholine receptors activated by carbachol, an acetylcholine analog. Thus, relief of G-protein inhibition appears to produce a novel form of short-term synaptic facilitation in cultured neurons. Similar short-term synaptic plasticity may be present at a wide variety of synapses, as it could occur during autoreceptor inhibition by glutamate or GABA, heterosynaptic inhibition by GABA, tonic adenosine inhibition, and in many other instances.

N-type calcium channel inactivation probed by gating-current analysis.

N-type calcium channels inactivate most rapidly in response to moderate, not extreme depolarization. This behavior reflects an inactivation rate that bears a U-shaped dependence on voltage. Despite this apparent similarity to calcium-dependent inactivation, N-type channel inactivation is insensitive to the identity of divalent charge carrier and, in some reports, to the level of internal buffering of divalent cations. Hence, the inactivation of N-type channels fits poorly with the "classic" profile for either voltage-dependent or calcium-dependent inactivation. To investigate this unusual inactivation behavior, we expressed recombinant N-type calcium channels in mammalian HEK 293 cells, permitting in-depth correlation of ionic current inactivation with potential alterations of gating current properties. Such correlative measurements have been particularly useful in distinguishing among various inactivation mechanisms in other voltage-gated channels. Our main results are the following: 1) The degree of gating charge immobilization was unchanged by the block of ionic current and precisely matched by the extent of ionic current inactivation. These results argue for a purely voltage-dependent mechanism of inactivation. 2) The inactivation rate was fastest at a voltage where only approximately (1)/(3) of the total gating charge had moved. This unusual experimental finding implies that inactivation occurs most rapidly from intermediate closed conformations along the activation pathway, as we demonstrate with novel analytic arguments applied to coupled-inactivation schemes. These results provide strong, complementary support for a "preferential closed-state" inactivation mechanism, recently proposed on the basis of ionic current measurements of recombinant N-type channels (Patil et al., . Neuron. 20:1027-1038).

Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L-type calcium channels.

Elevated intracellular Ca2+ triggers inactivation of L-type calcium channels, providing negative Ca2+ feedback in many cells. Ca2+ binding to the main alpha1c channel subunit has been widely proposed to initiate such Ca2+ -dependent inactivation. Here, we find that overexpression of mutant, Ca2+ -insensitive calmodulin (CaM) ablates Ca2+ -dependent inactivation in a "dominant-negative" manner. This result demonstrates that CaM is the actual Ca2+ sensor for inactivation and suggests that CaM is constitutively tethered to the channel complex. Inactivation is likely to occur via Ca2+ -dependent interaction of tethered CaM with an IQ-like motif on the carboxyl tail of alpha1c. CaM also binds to analogous IQ regions of N-, P/Q-, and R-type calcium channels, suggesting that CaM-mediated effects may be widespread in the calcium channel family.

Mechanism of auxiliary subunit modulation of neuronal alpha1E calcium channels.

Voltage-gated calcium channels are composed of a main pore-forming alpha1 moiety, and one or more auxiliary subunits (beta, alpha2 delta) that modulate channel properties. Because modulatory properties may vary greatly with different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of alpha1E calcium channels in combination with a beta (beta1-beta4) and/or the alpha2 delta subunit, exploiting both ionic- and gating-current measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting alpha2delta with beta subunits, of transfecting two different beta subunits simultaneously, and of COOH-terminal truncation of alpha1E to remove a second beta binding site. The main results are as follows. (a) The alpha2delta and beta subunits modulate alpha1E in fundamentally different ways. The sole effect of alpha2 delta is to increase current density by elevating channel density. By contrast, though beta subunits also increase functional channel number, they also enhance maximum open probability (Gmax/Qmax) and hyperpolarize the voltage dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation kinetics. Different beta isoforms produce nearly indistinguishable effects on activation. However, beta subunits produced clear, isoform-specific effects on inactivation properties. (b) All the beta subunit effects can be explained by a gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no clear evidence for simultaneous modulation by two different beta subunits. (d) The modulatory features found here for alpha1E do not generalize uniformly to other alpha1 channel types, as alpha1C activation gating shows marked beta isoform dependence that is absent for alpha1E. Together, these results help to establish a more comprehensive picture of auxiliary-subunit regulation of alpha1E calcium channels.

Preferential closed-state inactivation of neuronal calcium channels.

We have investigated the inactivation mechanism of neuronal N-, P/Q-, and R-type calcium channels. Although channels inactivate slowly during square-pulse depolarization, as observed previously, we now find that they inactivate profoundly during a train of action potential (AP) waveforms. The apparent paradox arises from a voltage-dependent mechanism in which channels inactivate preferentially from intermediate closed states along the activation pathway. Inactivation can therefore extend beyond the brief duration of AP waveforms to continue between spikes, as the channel undergoes repetitive cycles of activation and deactivation. The extent of inactivation during a train is strongly affected by the subunit composition of channels. Preferential closed-state inactivation of neuronal calcium channels could produce widely variable depression of Ca2+ entry during a train of APs.

Inhibition of recombinant Ca2+ channels by benzothiazepines and phenylalkylamines: class-specific pharmacology and underlying molecular determinants.

To understand the molecular basis of state-dependent pharmacological blockade of voltage-gated Ca2+ channels, we systematically characterized phenylalkylamine and benzothiazepine inhibition of three molecular classes of Ca2+ channels (alpha1C, alpha1A, and alpha1E) expressed from cDNA clones transfected into HEK 293 cells. State-dependent blockade figures importantly in the therapeutically desirable property of use-dependent drug action. Verapamil (a phenylalkylamine) and diltiazem (a benzothiazepine) were imperfectly selective, so differences in the state dependence of inhibition could be compared among the various channels. We found only quantitative differences in pharmacological profile of verapamil: half-maximal inhibitory concentrations spanned a 2-fold range (70 microM for alpha1A, 100 microM for alpha1E, and 110 microM for alpha1C), and inhibition was state dependent in all channels. In contrast, diltiazem produced only state-dependent block of alpha1C channels; alpha1A and alpha1E channels demonstrated state-independent block despite similar half-maximal inhibitory concentrations (60 microM for alpha1C, 220 microM for alpha1E, and 270 microM for alpha1A). To explore the molecular basis for the sharp distinction in state-dependent inhibition by diltiazem, we constructed chimeric channels from alpha1C and alpha1A and localized the structural determinants for state dependence to repeats III and IV of alpha1C, which have been found to contain the structures required for benzothiazepine binding. We then constructed a mutant alpha1C construct by changing three amino acids in IVS6 (Y14901, A1494S, 11497M) that have been implicated as key coordinating sites for avid benzothiazepine binding. Although these mutations increased the half-maximal inhibitory concentration of diltiazem inhibition by approximately 10-fold, the state-dependent nature of inhibition was spared. This result points to the existence of physically distinct elements controlling drug binding and access to the binding site, thereby favoring a "guarded-receptor" rather than a "modulated-receptor" mechanism of drug inhibition.

Bursts of action potential waveforms relieve G-protein inhibition of recombinant P/Q-type Ca2+ channels in HEK 293 cells.

1. A variety of neurotransmitters act through G-protein-coupled receptors to decrease synaptic transmission, largely by inhibiting the voltage-gated calcium channels that trigger neurotransmitter release. However, these presynaptic calcium channels are typically inaccessible to electrophysiological characterization. We have reconstituted a part of this inhibition using recombinant P/Q-type calcium channels and M2 acetylcholine receptors in HEK 293 cells. 2. One of the most interesting features of G-protein inhibition of calcium channels is that strong step depolarization transiently relieves the inhibition. We have found that short bursts of action potential voltage waveforms can also relieve the inhibition, increasing calcium current through G-protein-inhibited channels but not through uninhibited channels. 3. The extent of this relief increased linearly with the duration of the action potential waveforms. 4. This result provides the strongest evidence to date favouring the possibility that relief of G-protein inhibition can occur during high frequency trains of action potentials. This effect may constitute a novel form of short-term synaptic plasticity that is sensitive to action potential timing and duration.

G-protein modulation of N-type calcium channel gating current in human embryonic kidney cells (HEK 293).

1. Voltage-dependent inhibition of N-type calcium currents by G-proteins contributes importantly to presynaptic inhibition. To examine the effect of G-proteins on key intermediary transitions leading to channel opening, we measured both gating and ionic currents arising from recombinant N-type channels (alpha 1B, beta 1b and alpha 2) expressed in transiently transfected human embryonic kidney cells (HEK 293). Recombinant expression of a homogeneous population of channels provided a favourable system for rigorous examination of the mechanisms underlying G-protein modulation. 2. During intracellular dialysis with GTP gamma S to activate G-proteins, ionic currents demonstrated classic features of voltage-dependent inhibition, i.e. strong depolarizing prepulses increased ionic currents and produced hyperpolarizing shifts in the voltage-dependent activation of ionic current. No such effects were observed with GDP beta S present to minimize G-protein activity. 3. Gating currents were clearly resolved after ionic current blockade with 0.1 mM free La3+, enabling this first report of gating charge translocation arising exclusively from N-type channels. G-proteins decreased the amplitude of gating currents and produced depolarizing shifts in the voltage-dependent activation of gating charge movement. However, the greatest effect was to induce a approximately 20 mV separation between the voltage-dependent activation of gating charge movement and ionic current. Strong depolarizing prepulses largely reversed these effects. These modulatory features provide telling clues about the kinetic steps affected by G-proteins because gating currents arise from the movement of voltage sensors that trigger channel activation. 4. The mechanistic implications of concomitant G-protein-mediated changes in gating and ionic currents are discussed. We argue that G-proteins act to inhibit both voltage-sensor movement and the transduction of voltage-sensor activation into channel opening.

Elementary events underlying voltage-dependent G-protein inhibition of N-type calcium channels.

Voltage-dependent G-protein inhibition of N-type calcium channels reduces presynaptic calcium entry, sharply attenuating neurotransmitter release. Studies in neurons demonstrate that G-proteins have multiple modulatory effects on N-type channels. The observed changes may reflect genuine complexity in G-protein action and/or the intricate interactions of multiple channels and receptors in neurons. Expression of recombinant M2-muscarinic receptors and N-type channels in HEK 293 cells allowed voltage-dependent inhibition to be studied in isolation. In this system, receptor-activated G-proteins had only one effect: a 10-fold increase in the time required for channels to first open following membrane depolarization. There were no changes in gating after the channel first opened, and unitary currents were not detectably altered by modulation. Despite its simplicity, this single change successfully accounts for the complex alterations in whole-cell current observed during G-protein inhibition in neurons.

The alpha 1E calcium channel exhibits permeation properties similar to low-voltage-activated calcium channels.

The physiological and pharmacological properties of the alpha 1E calcium (Ca) channel subtype do not exactly match any of the established categories described for native neuronal Ca currents. Many of the key diagnostic features used to assign cloned Ca channels to their native counterparts, however, are dependent on a number of factors, including cellular environment, beta subunit coexpression, and modulation by second messengers and G-proteins. Here, by examining the intrinsic pore characteristics of a family of transiently expressed neuronal Ca channels, we demonstrate that the permeation properties of alpha 1E closely resemble those described for a subset of low-threshold Ca channels. The alpha 1A (P-/Q-type), alpha 1B (N-type), and alpha 1C (L-type) high-threshold Ca channels all exhibit larger whole-cell currents with barium (Ba) as the charge carrier as compared with Ca or strontium (Sr). In contrast, macroscopic alpha 1E currents are largest in Sr, followed by Ca and then Ba. The unique permeation properties of alpha 1E are maintained at the single-channel level, are independent of the nature of the expression system, and are not affected by coexpression of alpha 2 and beta subunits. Overall, the permeation characteristics of alpha 1E are distinct from those described for R-type currents and share some similarities with native low-threshold Ca channels.