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RAPID REPORT |
subunit increase the Ca2+ permeability of the human endplate ACh receptor
1 Istituto Pasteur-Fondazione Cenci Bolognetti and Dipartimento di Fisiologia Umana e Farmacologia, Università
‘La Sapienza’ P.le A. Moro 5; I-00185 Roma, Italy
2 Neuromed, Istituto di Ricovero e Cura a Carattere Scientifico, Via Atinese 18; I-86077 Pozzilli, Italy
3 Istituto di Medicina e Scienza dello Sport, CONI Servizi, Roma, Italy
4 Muscle Research Laboratory, Mayo Clinic, Rochester, MN 55905, USA
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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subunit of the human endplate ACh receptor (AChR) is a key determinant of the large fraction of the ACh-evoked current carried by Ca2+ ions (Pf). Consequently, missense mutations in the subunit are potential targets for altering the Pf of human AChR. In this paper we investigate the effects of two pathogenic point mutations in the M2 transmembrane segment AChR subunit, T264P and V259F, that cause slow-channel syndromes (SCS). When expressed in GH4C1 cells, the mutant receptors subunits raise Ca2+ permeability of the receptors 
1.5 and 2-fold above that of wild-type, to attain Pf values of 11.8% (T264P) and 15.4% (V259F). The latter value exceeds most Pf values reported to date for ligand-gated ion channels. Consistent with these findings, the biionic Ca2+ permeability ratio (PCa/PCs) of the mutant AChRs is also increased. Upon repetitive stimulation with ACh, the mutant receptors show an enhanced current run-down compared with wild-type, leading to a strong reduction of their function. We propose that the enhanced Ca2+ permeability of the mutant receptors overrides the protective effect of desensitization and, together with the prolonged opening events of the AChR channel, is an important determinant of the excitotoxic endplate damage in the SCS.
(Received 11 January 2007;
accepted after revision 31 January 2007;
first published online 1 February 2007)
Corresponding author Correspondence: F. Grassi. Dipartimento di Fisiologia Umana e Farmacologia, Università
‘La Sapienza’ P.le A. Moro 5; I-00185 Roma, Italy. Email: francesca.grassi{at}uniroma1.it
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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subunit (V259F) that lines the channel pore.
Hyperphosphorylation of tau protein leads to formation of tubulofilamentous structures in neuronal diseases as well as in IBM, and is associated with, and possibly caused by, a disrupted Ca2+ homeostasis (LaFerla, 2002; Pierrot et al. 2004). We recently showed that the fractional Ca2+ current (Pf) of human endplate AChR, defined as the percentage of the total ACh-evoked current carried by Ca2+ ions, is twice as large than that of the homologous mouse AChR (Fucile et al. 2006). The high Ca2+ permeability of human AChR by itself enhances vulnerability of the human endplate to excitotoxic damage (endplate myopathy) in the SCS. An increased fractional Ca2+ current would further contribute to Ca2+ overloading of the postsynaptic region and to the Ca2+-dependent pathological effects. Since the human subunit turned out to be a key determinant of the high Pf at the human endplate (Fucile et al. 2006), we postulated that strategically positioned amino acid substitutions in the subunit can alter the Ca2+ permeability of the AChR channel.
To test this notion, we measured the Pf of the V259F-AChR and found it almost twice as high as that of wild-type (WT) AChR. To determine whether this effect is mutation specific, we also examined the Pf for another well-characterized SCS mutation in the M2 segment of the subunit, T264P (Ohno et al. 1995). We found that the Pf of the T264P-AChR is also markedly increased compared with that of the WT receptor. Our results provide the first direct evidence that mutations associated with SCS alter Ca2+ permeability as well as channel kinetics of the human endplate AChR.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Rat pituitary GH4C1 cells were grown (5% CO2, 37°C) in HAM F10 medium plus 10% fetal calf serum and 1% penicillin/streptomycin. Cells were plated onto 35 mm Petri dishes (3 x 105 cells per dish) 24 h prior to transfection. The cDNAs encoding human wild-type 
, 
, 
, subunits, or subunits harbouring the T264P or V259F mutations were transiently transfected using Lipofectamine 2000, using 1 µg of cDNA for each subunit per Petri dish. Medium was changed after overnight incubation with cDNAs, and experiments carried out after a further 24–48 h. All media were purchased from Invitrogen.
Solutions and chemicals
Cells were superfused with a standard external medium containing (mM): 140 NaCl, 2.8 KCl, 2 CaCl2, 2 MgCl2, 10 Hepes-NaOH, 10 glucose, pH 7.3. For cell-attached experiments, patch pipettes were filled with the same solution plus ACh (200 nM).
For whole-cell recordings, patch pipettes were filled with a solution containing (mM): 120 KCl, 5 BAPTA, 10 Hepes-KOH, 2 Mg-ATP, 2 MgCl2, pH 7.3.
For Pf determinations, intracellular solution contained (mM): 140 N-methyl-D-glucamine (NMDG), 10 Hepes-HCl, 0.25 Fura-2 pentapotassium salt, 0.001 thapsigargin, pH 7.3. Calibration measurements were performed using an extracellular solution made of (mM): 153 NMDG, 10 CaCl2, 10 Hepes-HCl, pH 7.3.
The shift of the reversal potential of ACh-evoked current was measured using an intracellular solution containing (mM): 140 CsCl, 10 Hepes-CsOH, 20 BAPTA, pH 7.3. The external solution contained (mM): 150 CsCl, 10 Hepes-CsOH, 10 glucose, pH 7.3 plus 1 or 10 CaCl2.
All chemicals were purchased from Sigma (USA), except for Fura-2 pentapotassium salt (Molecular Probes).
Electrophysiology
Currents were recorded using borosilicate glass patch pipettes (2–5 M
tip resistance, Sylgard-coated for single-channel recordings) and an Axopatch 200B amplifier (Molecular Devices, Union City, CA, USA). Data were recorded and analysed using pCLAMP 9 (Molecular Devices). All recordings were performed at 25–27°C.
For whole-cell recordings, series resistance was compensated by 80–90%. Cells were voltage clamped at a holding potential of –60 mV and continuously superfused using a gravity-driven fast exchanger perfusion system (RSC-200, BioLogique, France). Current decay and run-down were fitted with single exponential curves [i(t) =
i
+
i0e–t/
], using Origin 7 (OriginLab Corporation, Northampton, MA, USA).
The relative Ca2+ permeability in biionic conditions (PCa/PCs) was estimated from the shift of the reversal potential (Vrev) of whole-cell ACh-induced current using two different extracellular Ca2+ concentrations (Cao1 of 1 mM, Cao10 of 10 mM). In each cell, Vrev was calculated at both Ca2+ concentrations (Vr1 and Vr10, respectively), by linear interpolation of the ACh-evoked currents peak amplitude plotted versus the test potential. The use of voltage ramp yielded inaccurate results because of the rapid desensitization of V259F-AChR. PCa/PCs ratios were obtained from the extended Lewis equation adapted to the chosen ion concentrations (Lewis, 1979; Castro & Albuquerque, 1995):
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| (1) |
ACh dose–current curves were constructed applying to each cell different doses of transmitter (0.1–300 µM, applied at 30–60 s interval) and normalizing the current response to the plateau value. The data were best-fitted using Hill equation by Origin 7 software (Origin Laboratory).
Single-channel data were filtered at 5 kHz and sampled at 25 kHz. Analysis was performed with a 50% threshold criterion, omitting events shorter than 0.12 ms. Slope conductance was calculated by linear fitting of the unitary amplitudes recorded at different pipette potentials (at least three for each cell). The fit was extrapolated to estimate cell resting potential, which was summed to the pipette potential to obtain membrane potential. The critical time used to identify a burst, determined for each cell from the closed time distribution (Colquhoun & Sakmann, 1985), ranged between 1 and 4 ms.
All results are given as mean ± S.E.M. Two data sets were considered statistically different when P < 0.05 (ANOVA test).
Pf determination
The methods to measure Pf have been fully reported previously (Fucile et al. 2000, 2006). Fluorescence determinations were made using a fluorescence upright microscope (Axioskop 2, Zeiss, Germany), a digital 12-bit cooled camera (SensiCam, PCO, Germany) and a monochromator (Cairn, UK). The system was driven by Axon Imaging Workbench 2 software (Molecular Devices), which also triggered the start of electrophysiological recording. All optical parameters and the setting of the digital camera (50 ms exposure time, 4 x 4 binning) were maintained throughout all measurements. The changes in intracellular calcium were monitored at a single excitation wavelength (380 nm), to achieve a higher time resolution, and expressed as the ratio of time-resolved fluorescence variation over the basal fluorescence (
F/F0).
Only isolated cells with low basal intracellular Ca2+ (F340/F380 ratio values < 2) were considered for recordings. Cells were filled with Fura-2 pentapotassium salt through the patch pipette and measurements performed after obtaining a stable value of basal fluorescence, with F380 > 200 arbitrary units (a.u), indicating satisfactory loading. The ratio F/Q (expressed in nC–1) was defined as:
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| (2) |
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| (3) |
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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V259F and T264P AChRs in GH4C1 cells
We first determined the main functional characteristics of wild-type (WT) and mutant AChRs expressed in GH4C1 cells. We confirmed the slow-channel properties of both V259F- and T264P-AChR at the single-channel level by cell-attached recordings (Fig. 1A). For the WT-AChR, the mean single channel slope conductance was about 50 pS and the mean burst duration was 1.9 ms (Table 1). As shown in Fig. 1B (left), the distribution of burst duration was fitted by two exponential components, with the mean time constants (b1 and b2) and weights given in Table 1. The duration of the bursts was greatly enhanced by the V259F as well as the T264P mutation, while the conductance of unitary events was not significantly affected (Table 1). The distribution of burst durations presented a third exponential component (time constant, b3) for both mutant AChRs. Typical examples are shown in Fig. 1B (middle and right panels) and mean values of the bs reported in Table 1. These data confirm that both mutant receptors retain their main functional properties when expressed in GH4C1 cells.
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V259F-AChR and T264P-AChR, respectively (data not shown).
During sustained ACh application, whole-cell currents decayed exponentially, reflecting receptor desensitization. At the plateau concentration of 100 µM, the rate of decay was similar for the WT-AChR (decay
= 207 ± 28 ms, n
= 14) and the T264P-AChR (246 ± 37 ms, n
= 7; P
= 0.4), whereas the V259F-AChR showed a faster decay (128 ± 16 ms, n
= 10; P
= 0.04). Repetitive ACh applications produced currents with decreasing peak amplitude (current run-down) for all three AChRs examined (Fig. 1C). An exponential fit of the consecutive current peaks showed that WT-AChR (Fig. 1C, left) had a relatively slow and limited run-down (run-down
= 0.78 ± 0.08 s, n
= 14), with an asymptotic amplitude (i) of 31 ± 4% of the first response. For the V259F mutant (Fig. 1C, middle), the decay was both faster (run-down
= 0.38 ± 0.06 s, n
= 9, P
= 0.002) and more pronounced (i
= 4.5 ± 0.9%) than for WT-AChR. The T264P mutant AChR (Fig. 1C, right) showed an intermediate behaviour, with a run-down
= 0.68 ± 0.05 s (n
= 7) and an asymptote at i
= 9 ± 2%, significantly less than that of WT-AChR (P
= 0.001). Current run-down was not accompanied by an increased rate of desensitization, as, in each cell, the decay of the third current response was not significantly different from that of the first response (P > 0.5). Together, these data indicate that the mutant receptors become less responsive than WT, or unresponsive, to ACh at physiological rates of stimulation.
Pf and PCa/PCs measurements
For Pf determinations we used nicotine because ACh might activate muscarinic receptors, leading to Ca2+ release from internal stores. Currents evoked by nicotine (100 µM) were comparable to those elicited by ACh, except for a slower current decay observed for the WT-AChR (decay
= 649 ± 87 ms, n
= 14) (e.g. Fig. 2A).
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V259F-AChR (Fig. 2A, middle) was 15.4 ± 1.7% (n
= 12, P
= 0.0007), showing that this mutation doubles the Ca2+ permeability of the AChR-channel. For comparison, we measured the Pf of the T264P-AChR (Fig. 2A, right) and again found a significantly enhanced Ca2+ permeability (Pf
= 11.76 ± 0.91%, n
= 13, P
= 0.003). Thus, both slow-channel mutations in the M2 segment of the subunit enhance the Ca2+ permeability of human AChR.
To test by a different approach that the Ca2+ permeability of the mutant AChRs was enhanced compared with that of WT-AChR, we also assessed the relative Ca2+ permeability of each isoform by measuring its biionic permeability ratio PCa/PCs. The currents evoked by ACh (10 µM) were measured at five or more test potentials between –10 and +10 mV, bracketing the current reversal potential, at two different extracellular Ca2+ concentrations (1 mM and 10 mM) (Fig. 3A). With 1 mM extracellular Ca2+, the average values of the reversal potentials were not statistically different for the V259F-AChR (0.2 ± 0.6 mV, n
= 7) or the T264P-AChR (–0.5 ± 0.3 mV, n
= 10) versus the WT-AChR (0.1 ± 0.3 mV, n
= 8; P > 0.2). The higher extracellular Ca2+ concentration induced a right-shift of the reversal potential of each isoform (Fig. 3B). Using eqn (1) (see Methods), we obtained PCa/PCs
= 0.73 ± 0.07 (n
= 7) for the WT-AChR. The value of PCa/PCs obtained for the V259F-AChR (1.20 ± 0.20, n
= 5) was significantly larger than that of WT-AChR (P
= 0.038). A further increase over the WT-AChR value was observed in the PCa/PCs ratio determined for the T264P-AChR (1.35 ± 0.10, n
= 7, P
= 0.0007).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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subunit. The mutations examined, V259F and T264P, gave rise to a fractional Ca2+ current of 15% and 12%, respectively. These Pf values are among the highest known, being equivalent to, or higher than, those reported for the NMDA receptor (up to 11%, Burnashev et al. 1995; 13.5%, Jatzke et al. 2002), or the homomeric 7-AChR (11.4%, Fucile et al. 2003).
The enhanced Ca2+ permeability of the SCS mutants additionally increases the Ca2+ ingress into the junctional sarcoplasm that stems from the prolonged duration of the synaptic current and the intrinsically high Ca2+ permeability of human AChR and is thus directly relevant to the pathogenesis of the endplate myopathy associated with the observed mutations. An increased permeability to Ca2+ may also occur with SCS mutations in other AChR subunits, as an increase of PCa/PCs was observed in HEK 293 cells expressing mouse V299F-AChR, a mutant receptor designed after a recently identified mutation in a SCS patient (Navedo et al. 2006). Both V259F and T264P mutations yield a complex effect on receptor function. The affinity of the mutant receptors for ACh is increased by about one order of magnitude, but this increase is accompanied by (or possibly results in) a loss of receptor function upon repetitive stimulation at high ACh concentration, as well as an enhanced rate of desensitization in the case of the V259F-AChR. That both mutant AChRs show an increased run-down during repetitive exposure to ACh probably contributes to the abnormal fatigability on exertion observed in vivo. Interestingly, the propensity of the mutant receptors to become unresponsive could partially mitigate the adverse effects of the prolonged open time and increased Ca2+ permeability of the mutant channels, but not enough to protect the endplate from excitotoxic injury.
In the only patient carrying the V259F mutation identified to date (Fidzianska et al. 2005), the endplate myopathy was accompanied by the presence of tubulofilamentous inclusion bodies. The formation of these structures might be precipitated by the severe disruption of Ca2+ homeostasis caused by the concurrent prolongation of synaptic events and doubled Pf, consistent with the notion that an altered Ca2+ homeostasis enhances phosphorylation of tau protein and formation of fibrillary structures (LaFerla, 2002).
Several lines of evidence indicate that the subunit is a key determinant of the Ca2+ permeability of adult muscle AChR. The Pf is almost 2-fold higher in AChRs containing the rather than the 
subunit (Villarroel & Sakmann, 1996; Ragozzino et al. 1998). Moreover, the subunit is the only determinant of the high Ca2+ permeability of the human adult AChR (Fucile et al. 2006). That a valine-to-phenylalanine mutation in the pore-forming M2 segment of the subunit (V259F) markedly enhances Pf whereas mutation of the corresponding residue in the subunit (V249F) has no effect on Pf (Fucile et al. 2006), is also consistent with the important role of the subunit in governing the Ca2+ permeability of the human receptor. Another mutation in the M2 segment, T264P, also increases the Pf of the mutant receptor. Thus, the data presented here confirm that the subunit has a strong effect on the Ca2+ permeability of human endplate AChR.
In our experiments, the alterations in PCa/PCs values only partially mirrored those in the Pf. The rapid run-down of current generated by V259F-AChR during multiple applications of ACh limits the accuracy of the PCa/PCs measurements. Furthermore, constant field assumptions leading to eqn (1) may not hold under the actual experimental conditions, as previously reported (Vernino et al. 1994; Burnashev et al. 1995). For instance, studies that compare muscle and neuronal AChRs reveal a 7-fold difference in the PCa/PCs ration (Vernino et al. 1994), but only a 2-fold difference for the Pf values (Vernino et al. 1993). Thus, biionic permeability ratios often provide qualitative rather than quantitative estimates of the relative Ca2+ permeability of a given channel whereas Pf determinations require no a priori assumptions (Zhou & Neher, 1994) and thus yield a more reliable measure of the Ca2+ permeability of the AChR channel (Burnashev et al. 1995).
In conclusion, our studies provide further insight into the pathogenic mechanisms whereby single amino acid substitutions in AChR subunits cause disease, which results from the delicate balance between the deleterious effects of the increased Ca2+ permeability and the dampening of the synaptic response by run-down of the investigated SCS mutant receptors at physiological rates of stimulation, by loss of AChR from the degenerating folds, and by the altered endplate geometry.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Castro NG & Albuquerque EX (1995). -Bungarotoxin-sensitive hippocampal nicotinic receptor channel has a high calcium permeability. Biophys J 68, 516–524.
Colquhoun D & Sakmann B (1985). Fast events in single-channel current activated by acetylcholine and its analogues at the frog muscle end-plate. J Physiol 369, 501–557.
Fidzianska A, Ryniewicz B, Shen X-M & Engel AG (2005). IBM-type inclusions in a patient with slow-channel syndrome caused by a mutation in the AChR epsilon subunit. Neuromuscular Dis 15, 753–759.[CrossRef][Medline]
Fucile S, Palma E, Mileo AM, Miledi R & Eusebi F (2000). Human neuronal threonine-for-leucine-248 7 mutant nicotinic acetylcholine receptors are highly Ca2+ permeable. Proc Natl Acad Sci U S A 97, 3643–3648.
Fucile S, Renzi M, Lax P & Eusebi F (2003). Fractional Ca2+ current through human neuronal 7 nicotinic acetylcholine receptors. Cell Calcium 34, 205–209.[CrossRef][Medline]
Fucile S, Sucapane A, Grassi F, Eusebi F & Engel AG (2006). The human adult subtype ACh receptor has high Ca2+ permeability and predisposes to endplate Ca2+ overloading. J Physiol 573, 35–43.
Jatzke C, Watanabe J & Wollmuth LP (2002). Voltage and concentration dependence of Ca2+ permeability in recombinant glutamate receptor subunits. J Physiol 538, 25–39.
LaFerla FM (2002). Calcium dyshomeostasis and intracellular signaling in Alzheimer's disease. Nat Rev Neurosci 3, 862–872.[Medline]
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Pierrot N, Ghisdal P, Caumont AS & Octave JN (2004). Intraneuronal amyloid- 1-42 production triggered by sustained increase of cytosolic calcium concentration induces neuronal death. J Neurochem 88, 1140–1150.[CrossRef][Medline]
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Villarroel A & Sakmann B (1996). Calcium permeability increase of endplate channels in rat muscle during postnatal development. J Physiol 496, 331–338.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, 
, 
, WT-AChR, as indicated.