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Neuroscience
J Physiol 0.0 (2014) pp 1–15
α-Synuclein forms non-selective cation channels and stimulates ATP-sensitive potassium channels in hippocampal neurons Sergej L. Mironov Institute of Neuro- and Sensory Physiology, Georg-August-University, G¨ottingen, 37073, Germany
Key points
r In Parkinson’s disease, the protein α-synuclein (αS) is produced within neurons and also appears in the extracellular fluid.
The Journal of Physiology
r In this study in hippocampal neurons, αS formed non-selective cation channels with multiple levels of conductance and rectification depending on their insertion site.
r αS channels induced local spontaneous increases in intracellular Na+ and Ca2+ , depolarized
neurons, augmented bursting activity and stimulated the opening of ATP-sensitive K+ channels.
r Non-selective channels were also observed in neurons transfected with either wild-type or mutant A53T αS, and after extracellular application of these proteins.
r The properties of αS channels in neuronal membranes suggest that extracellular αS is more toxic than αS produced within neurons.
Abstract In Parkinson’s disease and several other neurodegenerative diseases, the protein α-synuclein (αS) is produced within neurons and accumulates in the extracellular fluid. Several mechanisms of αS action are proposed, one of which is the formation of cation-permeable pores that may mediate toxicity. αS induces non-selective cation channels in lipid bilayers, but whether this occurs in living neurons and which properties the channels possess have not yet been examined. In this study the properties of αS channels in dissociated hippocampal neurons are documented. In cell-attached recordings the incorporation of αS into membranes was driven by applied negative potentials. These channels exhibited multiple levels of conductance (30, 70 and 120 pS at −100 mV) and inward rectification. The persistent activity of αS channels induced local changes in intracellular Na+ and Ca2+ , depolarized neurons and augmented bursting activity. αS channels formed by adding αS to the intracellular membrane in inside-out patches exhibited outward rectification. αS channels were equally permeable to Na+ , K+ and Ca2+ . These channels were also observed in neurons transfected with wild-type or mutant A53T αS, and after extracellular application of wild-type or mutant A53T αS proteins. Opening of αS channels stimulated opening of ATP-sensitive K+ (KATP ) channels and did not interfere with the activity of delayed rectifier K+ channels. The properties of αS channels in neuronal membranes suggest stronger toxicity of extracellularly applied αS than intracellular αS. Enhancement of neuronal excitability and distortions in ion homeostasis may underlie the toxic effects of αS that can be dampened by KATP channels. (Resubmitted 15 July 2014; accepted after revision 30 September 2014; first published online 17 October 2014) Corresponding author S. L. Mironov: DFG-Center of Molecular Physiology of the Brain, Institute of Neuro- and Sensory Physiology, Georg-August-University, G¨ottingen 37073, Germany. Email: [email protected] Abbreviations Aβ, amyloid β; ACSF, artificial cerebrospinal fluid; AP, action potential; αS, α-synuclein; BLM, bilayer lipid membrane; FWHM, full width half-maximum of fluorescence; KATP , ATP-sensitive K+ channel; Kdr , delayed rectifier K+ channel; m–v, mean–variance; Popen , open state probability; PD, Parkinson’s disease; τopen , mean open time; τclosed , mean closed time; WT, wild-type. C 2014 The Author. The Journal of Physiology C 2014 The Physiological Society
DOI: 10.1113/jphysiol.2014.280974
Downloaded from J Physiol (jp.physoc.org) at California Digital Library on November 19, 2014
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S. L. Mironov
Introduction α-Synuclein (αS) is a 14 kDa protein with an N-terminal domain containing several lysine residues and a C-terminal domain rich in glutamate residues. αS is a normal cytoplasmic protein but is also found in the interstitial space (Cookson, 2005). αS can associate with cells extracellularly, first anchoring to and then entering the lipid bilayer via the N-terminal domain (Tamamizu et al. 2006). Physiological functions of αS are not yet elucidated but its time-dependent expression in nervous tissue suggests specific role(s) in development. αS is not a pathological product itself and is produced continuously by most cells in the body throughout life. Intracellularly, it regulates the size of the presynaptic vesicle pool in the brain and modulates synaptic transmission (Murphy et al. 2000). The pathological actions of αS have attracted much attention in the last decade, especially in the pathogenesis of Parkinson’s disease (PD). Aggregated insoluble αS is the main component of Lewy bodies, which are the primary pathological characteristic of PD. However, soluble monomers and oligomers of αS in the human brain – rather than insoluble plaques – actually better correlate with PD severity. Such soluble products may cause cognitive and motor deficits in animal models of PD. The formation of pore-like αS complexes is supported by observations of doughnut-shaped protein particles by electron microscopy and atomic force microscopy (Tsigelny et al. 2012) and membrane permeabilization of lipid bilayers by αS (Zakharov et al. 2007; Di Pasquale et al. 2010; Tosatto et al. 2012) which has also been reported to occur for amyloid β (Aβ) (Arispe et al. 1993; Kourie et al. 2001; Bahadi et al. 2003; van Rooijen et al. 2010). A few studies have documented the ability of Aβ to induce ion pores in natural membranes (Kawahara et al. 1997; Demuro et al. ´ 2011; Sepulveda et al. 2014). However, detailed electrophysiological characterization of the hypothesized αS ion channels in neurons is lacking. Moreover, the inordinately high conductances of single αS channels measured in bilayer lipid membranes (BLMs) to date suggest very acute effects on neuronal excitability and ion homeostasis, which have not been consistently observed. The present study aimed to examine whether αS can form ion channels in hippocampal neurons and which properties they possess. I found that αS formed non-selective cation channels whose properties depended upon whether αS was applied from the extracellular or intracellular side. The asymmetry of αS actions has to be taken into account in considering possible effects of extra- and intracellular αS in the brain. Pathological actions of extracellular αS may dominate because the voltage dependence of channels in this case favours overexcitability of neurons and subsequent distortions in Ca2+ and Na+ homeostasis. The persistent activity
J Physiol 0.0
of αS channels did not affect the opening of delayed rectifier K+ (Kdr ) channels, but it did stimulate the activity of ATP-sensitive K+ (KATP ) channels that can dampen extracellular αS actions by opposing depolarization. Characterization of ion pores formed by αS in living neurons is an important step to elucidate the mechanisms underlying αS effects in the brain in normal and pathological conditions. Methods Preparation of neuronal cultures and reagents used
All animals were housed, cared for and killed in accordance with the recommendations of the European Commission (No. L358, ISSN 0378-6978), and animal use protocols were approved by the Committee for Animal Research, G¨ottingen University. The animals were killed by decapitation and the fresh brains were used for preparation of isolated neurons. Cultures of hippocampal neurons were prepared from 2- to 4-day-old mice as described previously (Mironov, 1995). Briefly, hippocampi were dissected from the brain; the cells were dissociated and plated on coverslips. Patch-clamp recordings and imaging were performed in neurons after >1 week in culture. All salts, glibenclamide, 4-aminopyridine, ouabain, calcium, sodium and glutamate receptor blockers and human α-synuclein (wild-type and the PD-related A53T mutant form) were from Sigma (Deisenhofen, Germany). Acetomethoxy (AM) ester forms of fluo-3 and Na-Green were from Invitrogen (Darmstadt, Germany). Green fluorescent protein (GFP)-tagged wild-type (WT) α-synuclein or GFP-tagged A53T mutant αS plasmids were obtained from Addgene (Cambridge, MA, USA; deposited by David Rubinsztein). Cells exposed to the transfection agent Lipofectamine 2000 (Invitrogen) only were used as controls. The transfection efficiency and cellular morphology were observed at 24 h and experiments were performed 2–3 days after transfection, which was 2–3 days after preparation. Electrophysiology and imaging
Membrane currents were measured with an EPC-7 amplifier (ESF, Friedland, Germany). The patch electrodes had a resistance of approximately 4 M for all pipette solutions used. Recorded traces were filtered at 3 kHz (−3 dB), and digitized at 10 kHz. The current and potential are presented according to conventions for intracellular recordings. For data obtained in the cell-attached and inside-out modes, the signs of current and potential were inverted. The channel open state probability (Popen ) was normalized to the maximum number of opened channels within the patch from which recordings were made. C 2014 The Author. The Journal of Physiology C 2014 The Physiological Society
Downloaded from J Physiol (jp.physoc.org) at California Digital Library on November 19, 2014
J Physiol 0.0
α-Synuclein channels in hippocampal neurons
Coverslips containing hippocampal neurons were placed on the microscope stage in a chamber continuously superfused at 1 ml min−1 at 34°C with bath solution (artificial cerebrospinal fluid (ACSF)) containing (in mM): 136 NaCl, 5 KCl, 1.25 CaCl2 , 6 glucose, 10 Hepes, pH 7.4. In patch-clamp experiments the pipette solution contained 154 mM NaCl or KCl, 88 mM CaCl2 and 30 mM Tris buffer (pH 7.4). The measured osmolarity of solutions ranged from 305 to 315 mosmol l–1 . After establishment of a gigaseal, the bath solution was changed to K+ -enriched ACSF to set the membrane potential to zero. αS solutions were always prepared fresh, filtered, kept on ice during experiments and used on the same day. Loading and measurements of intracellular Ca2+ and Na+ were performed as described previously (Mironov, 1995; Mironov & Langohr, 2005). The neurons were visualized with a ×10 or ×40 objective lens of an upright microscope (Axioscope 2, Zeiss). The excitation light from a CoolLED (BFI Optilas, Puchheim, Germany) was attenuated to 30%. Fluo-3 and Na-Green signals were excited at 475 nm and the fluorescence was collected at 535 ± 15 nm. Images were captured by cooled CCD camera (BFI Optilas) using ANDOR software (500 × 500 pixels at 12 bit resolution). For analysis images were background subtracted and evaluated offline with MetaMorph software (Universal Imaging Corp., Downington, PA, USA) and custom-made software. The images were deconvoluted to improve radial resolution (Mironov & Symonchuk, 2006) which decreased the full width half-maximum (FWHM) value to 0.4 μm. In imaging experiments αS was added to ACSF containing 1 μM ω-conotoxin GVIA and 10 μM nitrendipine to block the voltage-dependent N- and L-type calcium channels, respectively, 1 μM tetrodotoxin to block the voltage-dependent sodium channels and 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 μM (2R)-amino-5-phosphonovaleric acid (APV) to block ionotropic glutamate receptors. Data analysis
αS channels showed multiple openings and I quantified their activity using the mean–variance (m–v) method (Patlak, 1993). Necessary data are easily derived from the experimental traces and contain sufficient information to extract the basic characteristics of channel opening with multiple states. Tests using the m–v method in simulations showed reliable estimates of both unitary current levels (in ), state probabilities (Pn ) and mean open and closed times (τopen and τclosed ), as described below. Single channel activity consists of step-like events that represent the opening and closing of the channels. When m and v values are calculated within a window that slides across the trace, the 1-dimensional current trace is trans-
3
formed into a 2-dimensional m–v plot. Its pixels are grouped in spots that depict the dwell times of specific states and transitions between them. For example, when the channel resides in one state, the variance v 0 and m defines the mean current in this state. When the sliding window includes transition to another state, both mean and variance increase. Counting such events gives the number of transitions between the pair of specific states and was used to calculate τopen and τclosed . I first tested how m–v analysis describes the activity of one channel. The traces in Fig. 1A were generated for different closed times. The ‘experimental’ activity was sampled at 3 kHz and the mean and variance were calculated using the sliding window with 3 ms duration. The steady states of the channel and transitions between them are represented in the form of half-circles and parabolic arcs, respectively (Fig. 1). The single channel current is determined by the distance between the two distributions with v 0 and respective areas are proportional to the open and closed state probabilities (Popen and Pclosed ). The top graph in Fig. 1A shows that the calculated values matched the expectations well Popen = τopen /(τopen + τclosed ) and Pclosed = 1 − Popen defined by the input parameters, τopen and τclosed . The number of transitions between the closed and open state is equal to N = Popen τopen and was used to determine τopen . The closed time is then obtained as τclosed = τopen (1/Popen − 1). Calculated open and closed times were in accord with input values (bottom graph in Fig. 1A). Next the opening of two identical channels was considered. The traces in Fig. 1B present simulations of channel activity made with fixed open time and variable closed time. Dwell times for multiple identical channels are determined by a binomial distribution (Hille, 2001). In the case of two channels the occupancies of the closed and two open states are (1 − p)2 , 2p(1 − p), p2 , respectively, where p = Popen is the open state probability. Dwell times were obtained from the relative areas of three distributions with v 0 and they reproduced pre-set values well (Fig. 1B, the upper graph). The open and closed times were determined from the number of transitions between the first and second levels of conductance and previously determined p. The bottom graph in Fig. 1B shows that the calculated time constants were close to the input values. Application of m–v analysis to the traces generated by three and four channels also correctly recovered input values. The method was also tested for multiple channels with different current levels and m–v analysis predicted Popen , τopen and τclosed values well. The algorithms were programmed on Turbo-Pascal 7.0 and used to analyse single-channel data containing multiple openings. In some cases before the analysis the baseline was flattened using custom-made routines.
C 2014 The Author. The Journal of Physiology C 2014 The Physiological Society
Downloaded from J Physiol (jp.physoc.org) at California Digital Library on November 19, 2014
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S. L. Mironov
The current through the open channel was presented as the product of the Boltzmann and Ohm functions i=
i −100 + e0.04V i +100 (VX − V) 1 + e0.04V
J Physiol 0.0
calculations were done using Excel software (Microsoft Office 2003) and Statview (version 5.0.1, SAS Inc., Cary, NC, USA).
(1)
where i−100 and i+100 are the currents at −100 and +100 mV, respectively, and VX is the reversal potential (in mV) for the main permeant cation X. Parameters of the Boltzmann function for all ions were the same. In the analysis of selectivity the Ohmic function (V – VX ) was replaced by the Goldman equation (Hille, 2001). Equation (1) describes inward rectification in the cell-attached patches. In order to describe the outward rectification of αS channels in inside-out patches the sign of the exponential factor was inverted. Statistics
Each experimental protocol was repeated for at least four different patches from which channel recordings were made. The number of experiments is given in the text. All data are presented as mean ± standard deviation. Significant differences were determined using the non-parametric Mann–Whitney U test. Statistical
Results Formation of αS channels in hippocampal neurons
To examine the effects of αS, hippocampal neurons were patched using pipettes filled with 50–100 nM αS. In 72 cell-attached patches no native voltage-sensitive single channel activity was observed in the voltage range from −100 to +100 mV. Only such patches were used to examine the properties of αS channels. The absence of intrinsic voltage-sensitive channel activity in Na+ and Ca2+ -based pipette solutions was probably due to their low density or inactivation by recording in K+ -enriched solution that nullified membrane potential. Some recordings made with K+ -based pipette solutions showed the presence of the delayed rectifier (Kdr ) or ATP-sensitive (KATP ) K+ channels (n = 12 for both). Such patches were subsequently used to examine possible cross-talk with αS channels (see below and Figs 6 and 7). The presence of αS in the patch pipette alone was not sufficient for the appearance of αS channel activity.
Figure 1. Mean–variance analysis of single channel properties A, the traces in the leftmost panel present simulations of single channel activity. The unitary outward current was set to 4 pA, the random noise amplitude was 1 pA, the mean open time was 6 ms and mean closed time varied from 3 to 192 ms as indicated. The mean–variance (m–v) plots were obtained from 10 s-long traces using a sliding window with 3 ms duration. Small half-circles in the plots have variance close to zero and depict the dwell times in the closed and open states. The parabolic arcs between them present transitions between the states. ‘Experimental’ and theoretical open and closed state probabilities (top) and times (bottom) are plotted in the two graphs. Open symbols indicate input parameters and filled symbols show calculated values. B, the traces present simulations of activity of two identical channels. Simulation parameters were the same as in A. The two graphs on the right show the ‘experimental’ and theoretical probabilities (top) and times (bottom) for the two open and closed states. The open symbols indicate input parameters and filled symbols show their estimates obtained from m–v plots. C 2014 The Author. The Journal of Physiology C 2014 The Physiological Society
Downloaded from J Physiol (jp.physoc.org) at California Digital Library on November 19, 2014
α-Synuclein channels in hippocampal neurons
J Physiol 0.0
When initially silent patches were held at 0 mV for 30 min, αS channels were not formed; no opening was observed between voltages of −100 to +100 mV (n = 6, Fig. 2A, top). When the holding potential was shifted to −100 mV, αS channel opening became apparent (mean time of appearance 4.3 ± 0.4 min; n = 36; Fig. 2A, middle panel). Once channel activity was established, its pattern did not change for up to 60 min. Smaller concentrations of αS (down to 1 nM) also caused the formation of active channels. The channel formation time reciprocally decreased with increasing αS concentration. Because of the limited lifetime of patches, 30–100 nM αS was routinely
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used in experiments. Heat-inactivated αS was used as a control and no channel activity could be recorded in this case (n = 12). To examine the properties of single channels, inside-out patches were obtained immediately after formation of a gigaseal and 100 nM αS was then applied to the inner membrane side of the patch. In initially ‘silent’ patches no channel activity was evident in the range of voltages from −100 to +100 mV when they were held at 0 mV (n = 6; Fig. 2B, top). When it was shifted to −100 mV, αS channels appeared (the mean time for appearance of channel activity was 3.6 ± 0.5 min; n = 12; Fig. 2B). In cell-attached
Figure 2. α-Synuclein channels in hippocampal neurons All traces in A and B represent membrane current measured during voltage ramps from −100 to +100 mV. A, in cell-attached recordings the pipettes contained 30 nM α-synuclein in ACSF (the main permeating cation was Na+ , 136 mM). When the patch was held at 0 mV for 20 min no single channel activity was evident in the voltage range from −100 to +100 mV (three top traces). When the potential in this ‘silent’ patch was shifted to −100 mV, channel activity appeared within 4 min. B, application of 30 nM αS to the inner side of initially ‘silent’ patches induced channel activity after the holding potential was shifted to +100 mV for 5 min. The gating patterns in A and B were similar but the I–V curves had different rectification type and conductance levels. The insets demonstrate typical openings consisting of three different current steps. The lines represent the best fittings of I–V curves with eqn (1) used to describe rectification. Slope conductances of the channel at ±100 mV for the three conductance states are listed at opposite ends of the I–V curves. C, mean–variance plots for αS channels at different patch holding potentials obtained from the data shown in A and B. Smooth curves depict histograms of dwell times (probabilities of states) and were obtained by counting the pixels in m–v plots that have the same current amplitude and variance
Neuroscience
J Physiol 0.0 (2014) pp 1–15
α-Synuclein forms non-selective cation channels and stimulates ATP-sensitive potassium channels in hippocampal neurons Sergej L. Mironov Institute of Neuro- and Sensory Physiology, Georg-August-University, G¨ottingen, 37073, Germany
Key points
r In Parkinson’s disease, the protein α-synuclein (αS) is produced within neurons and also appears in the extracellular fluid.
The Journal of Physiology
r In this study in hippocampal neurons, αS formed non-selective cation channels with multiple levels of conductance and rectification depending on their insertion site.
r αS channels induced local spontaneous increases in intracellular Na+ and Ca2+ , depolarized
neurons, augmented bursting activity and stimulated the opening of ATP-sensitive K+ channels.
r Non-selective channels were also observed in neurons transfected with either wild-type or mutant A53T αS, and after extracellular application of these proteins.
r The properties of αS channels in neuronal membranes suggest that extracellular αS is more toxic than αS produced within neurons.
Abstract In Parkinson’s disease and several other neurodegenerative diseases, the protein α-synuclein (αS) is produced within neurons and accumulates in the extracellular fluid. Several mechanisms of αS action are proposed, one of which is the formation of cation-permeable pores that may mediate toxicity. αS induces non-selective cation channels in lipid bilayers, but whether this occurs in living neurons and which properties the channels possess have not yet been examined. In this study the properties of αS channels in dissociated hippocampal neurons are documented. In cell-attached recordings the incorporation of αS into membranes was driven by applied negative potentials. These channels exhibited multiple levels of conductance (30, 70 and 120 pS at −100 mV) and inward rectification. The persistent activity of αS channels induced local changes in intracellular Na+ and Ca2+ , depolarized neurons and augmented bursting activity. αS channels formed by adding αS to the intracellular membrane in inside-out patches exhibited outward rectification. αS channels were equally permeable to Na+ , K+ and Ca2+ . These channels were also observed in neurons transfected with wild-type or mutant A53T αS, and after extracellular application of wild-type or mutant A53T αS proteins. Opening of αS channels stimulated opening of ATP-sensitive K+ (KATP ) channels and did not interfere with the activity of delayed rectifier K+ channels. The properties of αS channels in neuronal membranes suggest stronger toxicity of extracellularly applied αS than intracellular αS. Enhancement of neuronal excitability and distortions in ion homeostasis may underlie the toxic effects of αS that can be dampened by KATP channels. (Resubmitted 15 July 2014; accepted after revision 30 September 2014; first published online 17 October 2014) Corresponding author S. L. Mironov: DFG-Center of Molecular Physiology of the Brain, Institute of Neuro- and Sensory Physiology, Georg-August-University, G¨ottingen 37073, Germany. Email: [email protected] Abbreviations Aβ, amyloid β; ACSF, artificial cerebrospinal fluid; AP, action potential; αS, α-synuclein; BLM, bilayer lipid membrane; FWHM, full width half-maximum of fluorescence; KATP , ATP-sensitive K+ channel; Kdr , delayed rectifier K+ channel; m–v, mean–variance; Popen , open state probability; PD, Parkinson’s disease; τopen , mean open time; τclosed , mean closed time; WT, wild-type. C 2014 The Author. The Journal of Physiology C 2014 The Physiological Society
DOI: 10.1113/jphysiol.2014.280974
Downloaded from J Physiol (jp.physoc.org) at California Digital Library on November 19, 2014
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S. L. Mironov
Introduction α-Synuclein (αS) is a 14 kDa protein with an N-terminal domain containing several lysine residues and a C-terminal domain rich in glutamate residues. αS is a normal cytoplasmic protein but is also found in the interstitial space (Cookson, 2005). αS can associate with cells extracellularly, first anchoring to and then entering the lipid bilayer via the N-terminal domain (Tamamizu et al. 2006). Physiological functions of αS are not yet elucidated but its time-dependent expression in nervous tissue suggests specific role(s) in development. αS is not a pathological product itself and is produced continuously by most cells in the body throughout life. Intracellularly, it regulates the size of the presynaptic vesicle pool in the brain and modulates synaptic transmission (Murphy et al. 2000). The pathological actions of αS have attracted much attention in the last decade, especially in the pathogenesis of Parkinson’s disease (PD). Aggregated insoluble αS is the main component of Lewy bodies, which are the primary pathological characteristic of PD. However, soluble monomers and oligomers of αS in the human brain – rather than insoluble plaques – actually better correlate with PD severity. Such soluble products may cause cognitive and motor deficits in animal models of PD. The formation of pore-like αS complexes is supported by observations of doughnut-shaped protein particles by electron microscopy and atomic force microscopy (Tsigelny et al. 2012) and membrane permeabilization of lipid bilayers by αS (Zakharov et al. 2007; Di Pasquale et al. 2010; Tosatto et al. 2012) which has also been reported to occur for amyloid β (Aβ) (Arispe et al. 1993; Kourie et al. 2001; Bahadi et al. 2003; van Rooijen et al. 2010). A few studies have documented the ability of Aβ to induce ion pores in natural membranes (Kawahara et al. 1997; Demuro et al. ´ 2011; Sepulveda et al. 2014). However, detailed electrophysiological characterization of the hypothesized αS ion channels in neurons is lacking. Moreover, the inordinately high conductances of single αS channels measured in bilayer lipid membranes (BLMs) to date suggest very acute effects on neuronal excitability and ion homeostasis, which have not been consistently observed. The present study aimed to examine whether αS can form ion channels in hippocampal neurons and which properties they possess. I found that αS formed non-selective cation channels whose properties depended upon whether αS was applied from the extracellular or intracellular side. The asymmetry of αS actions has to be taken into account in considering possible effects of extra- and intracellular αS in the brain. Pathological actions of extracellular αS may dominate because the voltage dependence of channels in this case favours overexcitability of neurons and subsequent distortions in Ca2+ and Na+ homeostasis. The persistent activity
J Physiol 0.0
of αS channels did not affect the opening of delayed rectifier K+ (Kdr ) channels, but it did stimulate the activity of ATP-sensitive K+ (KATP ) channels that can dampen extracellular αS actions by opposing depolarization. Characterization of ion pores formed by αS in living neurons is an important step to elucidate the mechanisms underlying αS effects in the brain in normal and pathological conditions. Methods Preparation of neuronal cultures and reagents used
All animals were housed, cared for and killed in accordance with the recommendations of the European Commission (No. L358, ISSN 0378-6978), and animal use protocols were approved by the Committee for Animal Research, G¨ottingen University. The animals were killed by decapitation and the fresh brains were used for preparation of isolated neurons. Cultures of hippocampal neurons were prepared from 2- to 4-day-old mice as described previously (Mironov, 1995). Briefly, hippocampi were dissected from the brain; the cells were dissociated and plated on coverslips. Patch-clamp recordings and imaging were performed in neurons after >1 week in culture. All salts, glibenclamide, 4-aminopyridine, ouabain, calcium, sodium and glutamate receptor blockers and human α-synuclein (wild-type and the PD-related A53T mutant form) were from Sigma (Deisenhofen, Germany). Acetomethoxy (AM) ester forms of fluo-3 and Na-Green were from Invitrogen (Darmstadt, Germany). Green fluorescent protein (GFP)-tagged wild-type (WT) α-synuclein or GFP-tagged A53T mutant αS plasmids were obtained from Addgene (Cambridge, MA, USA; deposited by David Rubinsztein). Cells exposed to the transfection agent Lipofectamine 2000 (Invitrogen) only were used as controls. The transfection efficiency and cellular morphology were observed at 24 h and experiments were performed 2–3 days after transfection, which was 2–3 days after preparation. Electrophysiology and imaging
Membrane currents were measured with an EPC-7 amplifier (ESF, Friedland, Germany). The patch electrodes had a resistance of approximately 4 M for all pipette solutions used. Recorded traces were filtered at 3 kHz (−3 dB), and digitized at 10 kHz. The current and potential are presented according to conventions for intracellular recordings. For data obtained in the cell-attached and inside-out modes, the signs of current and potential were inverted. The channel open state probability (Popen ) was normalized to the maximum number of opened channels within the patch from which recordings were made. C 2014 The Author. The Journal of Physiology C 2014 The Physiological Society
Downloaded from J Physiol (jp.physoc.org) at California Digital Library on November 19, 2014
J Physiol 0.0
α-Synuclein channels in hippocampal neurons
Coverslips containing hippocampal neurons were placed on the microscope stage in a chamber continuously superfused at 1 ml min−1 at 34°C with bath solution (artificial cerebrospinal fluid (ACSF)) containing (in mM): 136 NaCl, 5 KCl, 1.25 CaCl2 , 6 glucose, 10 Hepes, pH 7.4. In patch-clamp experiments the pipette solution contained 154 mM NaCl or KCl, 88 mM CaCl2 and 30 mM Tris buffer (pH 7.4). The measured osmolarity of solutions ranged from 305 to 315 mosmol l–1 . After establishment of a gigaseal, the bath solution was changed to K+ -enriched ACSF to set the membrane potential to zero. αS solutions were always prepared fresh, filtered, kept on ice during experiments and used on the same day. Loading and measurements of intracellular Ca2+ and Na+ were performed as described previously (Mironov, 1995; Mironov & Langohr, 2005). The neurons were visualized with a ×10 or ×40 objective lens of an upright microscope (Axioscope 2, Zeiss). The excitation light from a CoolLED (BFI Optilas, Puchheim, Germany) was attenuated to 30%. Fluo-3 and Na-Green signals were excited at 475 nm and the fluorescence was collected at 535 ± 15 nm. Images were captured by cooled CCD camera (BFI Optilas) using ANDOR software (500 × 500 pixels at 12 bit resolution). For analysis images were background subtracted and evaluated offline with MetaMorph software (Universal Imaging Corp., Downington, PA, USA) and custom-made software. The images were deconvoluted to improve radial resolution (Mironov & Symonchuk, 2006) which decreased the full width half-maximum (FWHM) value to 0.4 μm. In imaging experiments αS was added to ACSF containing 1 μM ω-conotoxin GVIA and 10 μM nitrendipine to block the voltage-dependent N- and L-type calcium channels, respectively, 1 μM tetrodotoxin to block the voltage-dependent sodium channels and 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 μM (2R)-amino-5-phosphonovaleric acid (APV) to block ionotropic glutamate receptors. Data analysis
αS channels showed multiple openings and I quantified their activity using the mean–variance (m–v) method (Patlak, 1993). Necessary data are easily derived from the experimental traces and contain sufficient information to extract the basic characteristics of channel opening with multiple states. Tests using the m–v method in simulations showed reliable estimates of both unitary current levels (in ), state probabilities (Pn ) and mean open and closed times (τopen and τclosed ), as described below. Single channel activity consists of step-like events that represent the opening and closing of the channels. When m and v values are calculated within a window that slides across the trace, the 1-dimensional current trace is trans-
3
formed into a 2-dimensional m–v plot. Its pixels are grouped in spots that depict the dwell times of specific states and transitions between them. For example, when the channel resides in one state, the variance v 0 and m defines the mean current in this state. When the sliding window includes transition to another state, both mean and variance increase. Counting such events gives the number of transitions between the pair of specific states and was used to calculate τopen and τclosed . I first tested how m–v analysis describes the activity of one channel. The traces in Fig. 1A were generated for different closed times. The ‘experimental’ activity was sampled at 3 kHz and the mean and variance were calculated using the sliding window with 3 ms duration. The steady states of the channel and transitions between them are represented in the form of half-circles and parabolic arcs, respectively (Fig. 1). The single channel current is determined by the distance between the two distributions with v 0 and respective areas are proportional to the open and closed state probabilities (Popen and Pclosed ). The top graph in Fig. 1A shows that the calculated values matched the expectations well Popen = τopen /(τopen + τclosed ) and Pclosed = 1 − Popen defined by the input parameters, τopen and τclosed . The number of transitions between the closed and open state is equal to N = Popen τopen and was used to determine τopen . The closed time is then obtained as τclosed = τopen (1/Popen − 1). Calculated open and closed times were in accord with input values (bottom graph in Fig. 1A). Next the opening of two identical channels was considered. The traces in Fig. 1B present simulations of channel activity made with fixed open time and variable closed time. Dwell times for multiple identical channels are determined by a binomial distribution (Hille, 2001). In the case of two channels the occupancies of the closed and two open states are (1 − p)2 , 2p(1 − p), p2 , respectively, where p = Popen is the open state probability. Dwell times were obtained from the relative areas of three distributions with v 0 and they reproduced pre-set values well (Fig. 1B, the upper graph). The open and closed times were determined from the number of transitions between the first and second levels of conductance and previously determined p. The bottom graph in Fig. 1B shows that the calculated time constants were close to the input values. Application of m–v analysis to the traces generated by three and four channels also correctly recovered input values. The method was also tested for multiple channels with different current levels and m–v analysis predicted Popen , τopen and τclosed values well. The algorithms were programmed on Turbo-Pascal 7.0 and used to analyse single-channel data containing multiple openings. In some cases before the analysis the baseline was flattened using custom-made routines.
C 2014 The Author. The Journal of Physiology C 2014 The Physiological Society
Downloaded from J Physiol (jp.physoc.org) at California Digital Library on November 19, 2014
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S. L. Mironov
The current through the open channel was presented as the product of the Boltzmann and Ohm functions i=
i −100 + e0.04V i +100 (VX − V) 1 + e0.04V
J Physiol 0.0
calculations were done using Excel software (Microsoft Office 2003) and Statview (version 5.0.1, SAS Inc., Cary, NC, USA).
(1)
where i−100 and i+100 are the currents at −100 and +100 mV, respectively, and VX is the reversal potential (in mV) for the main permeant cation X. Parameters of the Boltzmann function for all ions were the same. In the analysis of selectivity the Ohmic function (V – VX ) was replaced by the Goldman equation (Hille, 2001). Equation (1) describes inward rectification in the cell-attached patches. In order to describe the outward rectification of αS channels in inside-out patches the sign of the exponential factor was inverted. Statistics
Each experimental protocol was repeated for at least four different patches from which channel recordings were made. The number of experiments is given in the text. All data are presented as mean ± standard deviation. Significant differences were determined using the non-parametric Mann–Whitney U test. Statistical
Results Formation of αS channels in hippocampal neurons
To examine the effects of αS, hippocampal neurons were patched using pipettes filled with 50–100 nM αS. In 72 cell-attached patches no native voltage-sensitive single channel activity was observed in the voltage range from −100 to +100 mV. Only such patches were used to examine the properties of αS channels. The absence of intrinsic voltage-sensitive channel activity in Na+ and Ca2+ -based pipette solutions was probably due to their low density or inactivation by recording in K+ -enriched solution that nullified membrane potential. Some recordings made with K+ -based pipette solutions showed the presence of the delayed rectifier (Kdr ) or ATP-sensitive (KATP ) K+ channels (n = 12 for both). Such patches were subsequently used to examine possible cross-talk with αS channels (see below and Figs 6 and 7). The presence of αS in the patch pipette alone was not sufficient for the appearance of αS channel activity.
Figure 1. Mean–variance analysis of single channel properties A, the traces in the leftmost panel present simulations of single channel activity. The unitary outward current was set to 4 pA, the random noise amplitude was 1 pA, the mean open time was 6 ms and mean closed time varied from 3 to 192 ms as indicated. The mean–variance (m–v) plots were obtained from 10 s-long traces using a sliding window with 3 ms duration. Small half-circles in the plots have variance close to zero and depict the dwell times in the closed and open states. The parabolic arcs between them present transitions between the states. ‘Experimental’ and theoretical open and closed state probabilities (top) and times (bottom) are plotted in the two graphs. Open symbols indicate input parameters and filled symbols show calculated values. B, the traces present simulations of activity of two identical channels. Simulation parameters were the same as in A. The two graphs on the right show the ‘experimental’ and theoretical probabilities (top) and times (bottom) for the two open and closed states. The open symbols indicate input parameters and filled symbols show their estimates obtained from m–v plots. C 2014 The Author. The Journal of Physiology C 2014 The Physiological Society
Downloaded from J Physiol (jp.physoc.org) at California Digital Library on November 19, 2014
α-Synuclein channels in hippocampal neurons
J Physiol 0.0
When initially silent patches were held at 0 mV for 30 min, αS channels were not formed; no opening was observed between voltages of −100 to +100 mV (n = 6, Fig. 2A, top). When the holding potential was shifted to −100 mV, αS channel opening became apparent (mean time of appearance 4.3 ± 0.4 min; n = 36; Fig. 2A, middle panel). Once channel activity was established, its pattern did not change for up to 60 min. Smaller concentrations of αS (down to 1 nM) also caused the formation of active channels. The channel formation time reciprocally decreased with increasing αS concentration. Because of the limited lifetime of patches, 30–100 nM αS was routinely
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used in experiments. Heat-inactivated αS was used as a control and no channel activity could be recorded in this case (n = 12). To examine the properties of single channels, inside-out patches were obtained immediately after formation of a gigaseal and 100 nM αS was then applied to the inner membrane side of the patch. In initially ‘silent’ patches no channel activity was evident in the range of voltages from −100 to +100 mV when they were held at 0 mV (n = 6; Fig. 2B, top). When it was shifted to −100 mV, αS channels appeared (the mean time for appearance of channel activity was 3.6 ± 0.5 min; n = 12; Fig. 2B). In cell-attached
Figure 2. α-Synuclein channels in hippocampal neurons All traces in A and B represent membrane current measured during voltage ramps from −100 to +100 mV. A, in cell-attached recordings the pipettes contained 30 nM α-synuclein in ACSF (the main permeating cation was Na+ , 136 mM). When the patch was held at 0 mV for 20 min no single channel activity was evident in the voltage range from −100 to +100 mV (three top traces). When the potential in this ‘silent’ patch was shifted to −100 mV, channel activity appeared within 4 min. B, application of 30 nM αS to the inner side of initially ‘silent’ patches induced channel activity after the holding potential was shifted to +100 mV for 5 min. The gating patterns in A and B were similar but the I–V curves had different rectification type and conductance levels. The insets demonstrate typical openings consisting of three different current steps. The lines represent the best fittings of I–V curves with eqn (1) used to describe rectification. Slope conductances of the channel at ±100 mV for the three conductance states are listed at opposite ends of the I–V curves. C, mean–variance plots for αS channels at different patch holding potentials obtained from the data shown in A and B. Smooth curves depict histograms of dwell times (probabilities of states) and were obtained by counting the pixels in m–v plots that have the same current amplitude and variance
