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A simple method for fast temperature changes and its application to thermal activation of TRPV1 ion channels. León D. Islas*†, Victor De-la-Rosa*, Beatriz Rodríguez-Cortés*, Gisela E. Rangel-Yescas* and David Elias-Viñas°
*Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), México City, México, 04510 °Sección de Bioelectrónica, CINVESTAV-IPN, México City, México, 07360 †Correspondence to L.D.I. at:
[email protected]
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Abstract -Background
Thermally activated ion channels function as molecular thermometers and participate in other physiological important functions. The mechanism by which they acquire their exquisite temperature sensitivity is unknown and is currently an area of intense research. For this reason, there is a need for diverse methods to deliver controlled temperature stimuli. -New Method
We have developed a simple, inexpensive and reliable method to deliver temperature pulses to small volumes surrounding the recording area, which can be either a patchclamp pipette containing a cell-free membrane with thermally activated channels or a whole cell attached to a pipette. -Results
Here we developed a micro-heater based on resistive heating of a copper filament enclosed in a glass capillary that is capable of delivering fast and localized temperature changes. We validated the performance of the micro-heaters by analyzing the heatinduced activation of TRPV1 thermoTRP channels recorded in inside-out patches and demonstrate the use of the micro-heaters. -Comparison with Existing Method(s)
The micro-heaters we introduce here are compact, easy to fabricate and to operate. In contrast with bulk solution heaters commercially available, our method is extremely affordable and simple to operate. To the best of our knowledge there are no other similar, commercially available heating methods. -Conclusions
The micro-heater method is simple and should provide a straightforward and rapid experimental tool to study mechanisms in thermally activated ion channels. Key words TRPV1 channel, heat activation, thermoTRPs, resistive heating, patch-clamp, ion channels.
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Introduction Thermally activated ion channels are an important family of membrane proteins associated with sensory perception of temperature and nociceptive processes (Caterina et al., 1997; Everaerts et al., 2011). While some of these channels are activated by decreases in temperature (Peier et al., 2002; Brauchi et al., 2004), others open in response to increased temperature. The most important representatives of the later belong to the TRPV subfamily of TRP ion channels, which are found both in vertebrates and invertebrates. The most thoroughly characterized member of this subfamily is the TRPV1 cation-selective ion channel. This channel can be gated by very diverse chemical stimuli, opening in response to binding of capsaicin and the endogenous ligands LPA (Nieto-Posadas et al., 2011) and anandamide (Zygmunt et al., 1999). Perhaps more importantly, TRPV1 is directly activated by increases in temperature in the range of 40 oC to 50 oC (Caterina and Julius, 2001; Yao et al., 2010, 2011). Several other members of the TRPV subfamily are also activated by increases in temperature and still other related channels in invertebrates share the same property (Patapoutian et al., 2003). Although not directly activated by temperature, some ion channels not related to TRPs show very high temperature dependence, as demonstrated by Q10 coefficients of activation kinetics larger than ~5. Examples of these are the proton-permeable voltage dependent channels (Kuno et al., 2009; Fujiwara et al., 2012) and some chloride selective channel (Pusch et al., 1997).
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Studies of the thermal activation or modulation of activity of TRPV and other heat sensitive channels have to involve methods to change and control the surrounding temperature. The available methodologies vary from whole-bath temperature changes (Güler et al., 2002; Matta and Ahern, 2007), which due to the high thermal inertia of water are necessarily very slow, to infrared laser induced step changes in temperature in very small volumes (Yao et al., 2009), which is an ideal method, but in practice may be difficult to implement. Other solutions that have been put forward are the perfusion of heated solutions, which in certain cases can achieve sub-second changes in temperature, but also need a complicated implementation, often requiring fast piezodriven positional changers (Kuno et al., 2009). Several temperature gated ion channels show a marked desensitization process when exposed to elevated temperatures for prolonged periods of time (Everaerts et al., 2011). Some of the commercially available temperature controllers take too long to reach the prescribed temperature, aggravating the influence of this phenomenon. So, a general purpose, easy to implement and fast method of temperature change is desirable. It is also desirable that the temperature be changed in as small a volume as possible, in such a way that cooling also can occur rapidly. In this communication, we present an inexpensive, straightforward method that allows rapid temperature changes of an excised membrane patch or small cell under wholecell patch clamp. The method is easy to implement in an electrophysiology lab and provides a simple alternative to available methods of temperature control.
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Materials and Methods a. Design and construction of the micro-heater. The method we present here is based on resistive heating of a thin copper wire enclosed in a glass capillary. We use copper wire of 0.10 mm diameter (American Wire Gauge, AWG # 38-39), the kind that can be found in small voltage transformers. A small, approximately 15 cm long strand of copper wire should be cut and stripped of the insulating varnish, unless non-insulated wire is available.
This step is best
accomplished by passing a large (> 2 ampere) current through the wire, which should heat and burn off the varnish. The wire is then inserted into a glass capillary. We have used soft borosilicate hematocrit capillaries (VWR) and hard borosilicate capillaries for microelectrode fabrication (Warner Instruments) with identical results. Once the wire is in the capillary, this is pulled under an open flame from a Bunsen burner until it is the same diameter of the copper wire. Better results are accomplished with the use a portable butane gas torch, which produces a smaller flame. Pulling of the glass capillary should stop when the copper wire is in direct contact with the glass wall and can no longer be withdrawn from the capillary tube by pulling on it (Fig 1a). The drawn capillary is now bent under the open flame to form a hairpin (Fig. 1b). At this point, there should be about an inch of copper wire protruding from the capillary hairpin. A picture of the finished micro-heater is shown in Fig. 1 C. The wire ends are then soldered to insulated electrical wire, which should be terminated by banana-type end connectors. Any
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exposed wire should be insulated with heat-shrinking plastic tubes or heat resistant epoxy resin. The banana connectors are used to connect the micro-heater to a commercially available current-regulated DC power supply (Agilent). The micro-heater is attached to a manual micromanipulator for fine positioning in the recording chamber in front of the recording electrode (Fig 1d). The temperature surrounding the micro-heater can be recorded in several ways. We have used two methods; the first makes use of a commercially available small size thermistor (Warner Instruments) placed as close as possible to the micro-heater. This method is not as accurate because due to the size of the thermistor bead, the temperature it measures is an average of the temperature surrounding the micro-heater. The second method is based on recording the current through an open patch pipette placed right next to the recording electrode. This method takes advantage of the heat-dependence of the resistivity of electrolyte solutions (Lide and Kehiaian, 1994) and has the advantages that is rapid and can measure the actual temperature changes within microns of the patch-recording pipette. The temperature measuring pipettes can be calibrated before or after the experiment.
b. Experimental demonstration of micro-heater performance Heat-activated currents were recorded from inside-out patches obtained from HEK293 cells expressing rat TRPV1. HEK293 cells expressing large T-antigen were co-
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transfected with 400 ng of rTRPV1 in the pCDNA3 plasmid and 400 ng of the Green Fluorescent Protein (GFP) in the pIRES-GFP plasmid (BD Biosciences). Transfection was carried out with the reagent JetPei (Polyplus Transfection, France) using the manufacturer-suggested procedures. Green-fluorescing cells were identified as cells expressing TRPV1. Currents were recorded 24-48 hrs. after transfection with an Axopatch 200B patch clamp amplifier (Molecular Devices) and saved directly into the computer hard disc using an 18-bit analog to digital converter (Instrutech). Currents were filtered at 5 kHz and recorded at 15 kHz. The recording solutions were the same for the patch pipette and the bath and had the following composition (in mM): 130 NaCl, 5 HEPES, 2 KCl , 1 mM EGTA. Patch pipettes were drawn from thin wall borosilicate glass capillaries (Warner Instruments) using a horizontal pipette puller (Sutter Instruments) and fired polished to about 1-2 m diameter. Pipette resistance after polishing was 3-5 M. The same pipettes without polishing (~1.5 M resistance) were used for recording the openpipette current near an inside out patch, which is proportional to the temperature at the pipette tip. This current was recorded with a HEKA EPC 7 patch clamp amplifier (HEKA Electronik) and converted to temperature as follows. For each temperaturemeasuring pipette, a calibration curve was obtained after the experiment. The pipette was submerged in recording solution heated at different temperatures, which were measured with a thermistor (Warner Instruments). The resulting resistance vs. temperature calibration curve was used to convert resistance to temperature in the vicinity of the membrane patches. 7
After obtaining an inside-out patch, this was brought within 250 m of the surface of the micro-heater. All currents were recorded into a PC computer’s hard disk using Pulse software from HEKA. Data analysis was performed in Igor 6 (Wavemetrics) and simulations of the temperature distribution near the micro-heaters were carried out with programs written in Matlab (Mathworks). Whole-bath heating for comparison experiments was carried out with a small aluminum chamber heated by a Peltier devise controlled by an ALA Scientific heating and cooling module.
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Results In this paper we present a very simple and effective micro-heater that can quickly change the local temperature over a large range, between 22 to 55 degrees Celsius. Construction is straightforward and is described in the Methods section. Fig. 1 shows the general strategy of construction. A copper wire of approximately 100-micrometer diameter (American Wire Gauge (AWG) #38-39) is inserted into a glass capillary and this is drawn under an open flame so that the wire is in contact with the glass. Fig. 1c shows a picture of the final micro-heater. A bead of epoxy resin keeps the two branches together for increased stability of the glass micro-heater. Heating of the wire and glass is accomplished by passing a large electrical current. Fig. 2a shows the change in resistance of an electrode placed at ~250 m from the surface of the heater when the two are submerged in recording solution. Two consecutive current pulses were applied of the magnitude indicated in the figure inset. As can be seen, reproducible changes in resistance/temperature can be readily obtained near the heater. It is noteworthy that the local temperature returns to a value very close to the initial bath temperature (~23oC in this experiment) within a few seconds. In Fig. 2b, the resistance of the same electrode was calibrated in the same solution at different temperatures as measured by a thermistor. Resistance was measured at the end of the 15 sec current pulse. The relationship between electrical resistance and temperature is approximately linear and the deviation can be explained by the known temperaturedependence of the thermal conductivity of water and most other substances (Yusufova
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et al., 1975). Note that due to this curvature, a linear approximation to the resistancetemperature curve is not appropriate and each temperature-measuring pipette should be independently calibrated. The micro-heater can be used to deliver sequential changes in temperature, as illustrated in Fig. 3a. By sequentially varying the current passing through the wire, the heater increases the local temperature in a step-wise manner. The calibration curve for the pipette used in this experiment is shown in Fig. 3b and was obtained as in the experiment in Fig. 2. It is clear from this calibration that large temperature differentials can be readily achieved (>50oC). In order to further understand the temperature changes surrounding the heater, we carried out a simple numerical calculation by solving the heat equation in one dimension:
¶T ¶ 2T =k 2 ¶t ¶x
Eq. 1
This equation describes the temperature T (in oC) as a function of time, t and distance, x and does not take into account convective heat fluxes. Equation 1 was solved using a finite difference approximation implemented in a Matlab program, with the following initial and boundary conditions:
ì22 oC for 0 £ t £ 9 sec ï T ( x = ¥) = 22 oC; T ( x = 0) = í 70 oC for 9 £ t £ 18 sec ï o î22 C for 18 £ t £ 36 sec
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The thermal diffusivity, , of the saline solution was set to 0.08 x 10-6 m2/s which is slightly smaller than for pure water (0.14 x 10-6 m2/s), because the presence of electrolytes reduces the thermal conductivity of water (Yusufova et al., 1975). Results of the calculation are shown in Fig. 4a. Shown is the temperature distribution as a function of distance, x, for a 9 sec duration step change in temperature initiated at t = 9 sec and x = 0, which represents the surface of the micro-heater. As expected, the temperature decays quickly as the distance from the heater increases. Fig. 4b shows the time response of the temperature field at five distances from the surface of the heater. The temperature increases with a time constant that becomes larger as the distance increases, as expected. More interestingly, when we compare the calculated time course of the increase in temperature at 250m with the change in resistance of an open pipette placed ~250m away from the surface of the heater, it can be clearly seen that the simple heat diffusion model provides an accurate description of the temperature changes produced by the micro-heater (Fig. 4c). Having characterized the performance of the micro-heaters, we assess their functionality by carrying out temperature activation experiments in the TRPV1 heatactivated ion channel. Inside-out patches obtained from HEK cells transiently expressing rat TRPV1 channels were placed within 300 m of the micro-heater. The initial bath temperature was ~23oC, which is well below the threshold for heat activation of these channels (Yao et al., 2010). At this temperature, individual channel openings are visible on top of a leak current of about 50 pA (Fig 5a). When 2 A of current are delivered to the micro-heater, the current of a patch held at 60 mV 11
increases several-fold, and now a macroscopic current is recorded, indicating that the temperature at the patch increased well above 42oC, producing heat activation of the channels (Fig 5a). In Fig 5b we show currents elicited in response to repetitive voltage clamp pulses to 60 mV, at two different temperatures. Notice that after the heater is turned off, current through TRPV1 returns to its baseline value, as indicated by the fewer openings in trace c, Fig. 5a and implying that temperature returned to its initial value. Another example of the functioning of these micro-heaters is given in Fig. 6. Here consecutive current pulses of 1 sec duration and increasing amplitude are applied to a micro-heater placed near an inside-out patch. Control data from a non-transfected cell is shown in Fig. 6a and b. No currents are activated and the seal resistance decreases with increasing temperature. The temperature-dependence of the leak current has a Q10 of 1.4 (Fig 6b). Data from a TRPV1-expressin inside-out patch is shown in Fig. 6c and d. Since the channels are very rapidly activated (Yao et al., 2010), the current essentially follows the same kinetics as the temperature change. These experiments clearly show that the micro-heater is capable of producing fast activation of TRPV1 channels. Also show in Fig. 6d is the fact that after current through the micro-heater is turned off, the local temperature rapidly returns to a value near the bath bulk temperature, and the TRPV1 current also shuts-down quickly. A word of caution should be given here since after larger temperature changes, above 40oC, the heaters can affect the temperature of the
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bulk solution, effectively heating the whole bath. This problem can be dealt with if constant bath perfusion with a cooled solution is used during the experiment. Finally, we compared the performance of the micro-heaters with that of a whole-bath heater. Figure 7 shows that the change in magnitude of the currents activated at 60 mV by both methods is similar, with almost the same threshold and Q10 values, indicating that the micro-heaters do not distort temperature activation of TRPV1. Notice that, at least in this experiment, the micro-heater is able to reach a higher temperature.
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Discussion We heave constructed and tested a simple micro-heater capable of increasing the local temperature of electrolyte solutions approximately 30 degrees Celsius above the baseline temperature. The device is based on resistive heating of a copper filament enclosed in a thinned glass capillary and is extremely simple to build and operate. The total cost per micro-heater is less that a dollar. It can be rapidly turned on and off by controlling the current passing through the wire and its temperature is directly proportional to the magnitude of the current used to heat it. This micro-heater can be reliably used to study activation of TRPV1 heat-activated ion channels and has the added advantage that, since only very localized temperature changes are achieved, cooling is also very fast, which is a serious problem when other temperature changing devices are used, such as perfusion of heated solutions. These heaters should also find applications in studies of other channels where temperature needs to be locally controlled. Our theoretical calculations suggest that the temperature at the surface of the device rises almost instantly when current is switched on, because the temperature field in the solution surrounding the heater is accurately described by the solution to the heat equation for an instantaneous temperature pulse. This result suggests that the microheater could be driven with a power supply under some control scheme, such as pulsewidth modulation with feedback to increase the time response of temperature changes. Rapid control of the current delivered to the micro-heater can also be
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achieved by using programmable power supplies. We are currently working on developing a control circuit to deliver essentially instantaneous square pulses of temperature. As a final remark, this is not the first small-scale resistive heater device that has been proposed. Fred Sachs’ group presented a resistive heater the size of a patch pipette that could achieve very rapid local control of the temperature, but its fabrication is very involved and expensive (Auerbach et al., 1987). The advantage of our method is precisely that it can be implemented rapidly and at low cost.
Acknowledgments Research in L.D. Islas’s lab is supported by grant IN212612-3 from DGAPA-PAPIITUNAM and grant CB-151297 from CONACyT-México. V.D.R. was the recipient of a PhD scholarship from CONACyT (309408). We wish to acknowledge the expert technical assistance of Mr. Manuel Hernandez.
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Figure Legends
Figure 1. Schematic and pictures of the fabrication procedure and final microheater. The details of the micro-heater fabrication are given in the materials and methods. A) Schematic of the wire inserted in the capillary glass after been drawn and thinned under an open flame. B) After the step in A, the glass capillary is bent under heating to form a hairpin that acts as the surface of the micro-heater. C) Picture of a finished heater. Note the scale bar and the fact that the copper wire is in physical contact with the thinned capillary wall. D) The heater (lower part of the picture) placed in front of a patch-clamp recording electrode, imaged with a 20x objective. Figure 2. Performance of the micro-heater and calibration of temperature. A) An open electrode was placed in the recording chamber at a distance of ~250 from the surface of the micro-heater. The resistance of this electrode was recorded by measuring the current in response to fixed 60 mV pulses. A pulse of the indicated current magnitude was applied to the heater. This changes the conductivity of the solution in the vicinity of the electrode, changing the recorded resistance in a manner directly proportional to the temperature change. The increase in temperature occurs within seconds and it returns to the starting value also within few seconds. B) Calibration curve of the same electrode, obtained after the experiment. The absolute value of the temperature can be read directly from this graph.
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Figure 3. Sequential increases in temperature. Similar experiment as in Fig. 2, but the magnitude of the current applied to the heater was changed immediately after another. The pipette used to calibrate the temperature is different and its calibration curve is shown in panel B. Figure 4. Temperature distribution simulations. The heat equation (Eq.1) was numerically solved to give the temperature distribution as a function of distance and time. A) Distribution of temperature in the solution as a function of distance from the micro-heater surface in response to a 9 sec duration temperature step to 70 oC. B) Time course of temperature change at the indicated distances from the heater surface. Note that the time course is significantly different at different distances. C) Comparison of the simulation with experimental data. The change in resistance of the electrode used in the experiment in Fig. 2 in response to a 9 sec 3.5 amp current pulse is shown in black. The red trace is the time course of temperature change at 250 m (same as the yellow trace in Fig. 4b). The scale bar units of MΩ apply to the black resistance trace. Figure 5. Heat activation experiments in TRPV1. Inside–out patches were obtained from HEK cells expressing TRPV1 channels and placed near the micro-heater. A) Sequential pulses of the same current magnitude (2 Amp) where applied to elicit activation of the channels. Current was monitored by applying a double pulse voltage clamp protocol (inset in B) every 1 sec and the magnitude of the current at 60 mV plotted. Clear on-off responses to temperature can be easily obtained. B) An example of
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the current through TRPV1 channels in the patch in A at the basal temperature of ~23 oC
(a), with the heater on (b) and after turning the heater off (c).
Figure 6. Rapid and reversible heating. (A) Inside-out patch recordings from a nontransfected HEK cell at the indicated temperatures. Resistance of the patch decreases as the temperature increases, but no endogenous currents become activated. (B) Leak current magnitude increase in response to a ramp of temperature applied with a micro-heater. The Q10 of the leak current is 1.41. (C) An inside-out patch with TRPV1 channels was heated with the same micro-heater as usen in (A) via application of rapid (1 sec) sequential current pulses of different amplitude. Shown are current traces through TRPV1 channels at the indicated temperatures. (D) Time course of temperature and TRPV1 current monitored simultaneously. After the micro-heater current pulse is turned off, the temperature returns to a value near the bulk bath temperature, and the current through TRPV1 channels is also diminished as the channels turn off at the lower temperature. Notice that the activation kinetics are essentially the same as the kinetics of temperature change. Figure 7. Comparison with whole-bath heating. TRPV1 currents in inside-out patches were activated by the micro-heater method (solid circles) or by heating the solution in the recording chamber (hollow circles). The Q10’s are 6.64 and 5.81 for the currents activated by micro-heater or whole-bath heating, respectively.
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