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Plenary Article
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Piezo2 channel conductance and localization domains in Merkel cells of rat whisker hair follicles
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Ryo Ikeda a,b , Jianguo G. Gu a,∗ a
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Department of Anesthesiology, The University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0531, USA Department of Orthopaedic Surgery, Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan
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Keywords: Merkel cell Mechanotransduction Mechanoreceptor Piezo2 Whisker hair follicle
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1. Introduction
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We have recently shown that Merkel cells transduce tactile stimuli via Piezo2 channels to initiate the sense of touch. Here we performed patch-clamp recordings to assess single channel activity on the membranes of Merkel cells in whisker hair follicles. Under the cell-attached configuration, most Merkel cell membrane patches showed large outward unitary currents with single channel conductance being ∼200 pS. The outward unitary currents were not affected by negative pressures up to 150 mmHg when applied to the membrane patches. The application of negative pressures up to 190 mmHg also could not directly elicit any inward unitary current in the membrane patches. However, after establishing the whole-cell configuration, mechanically activated currents (MA) that resembled Piezo2 currents could be elicited by membrane displacements in every Merkel cell tested. While the MA current decayed rapidly, a small steady-state current component with significant channel noise could be observed. Applications of stationary and non-stationary fluctuation analyses to the MA currents yielded single channel conductance of 32.5 ± 3.8 and 54.0 ± 5.3 pS, respectively. The lack of mechanical responses under the cell-attached configuration and the existence of Piezo2 MA currents under the whole-cell configuration raised a possibility that Piezo2 channels are preferentially located on Merkel cell processes, the membrane domains inaccessible by recording electrodes. © 2014 Published by Elsevier Ireland Ltd.
Mammals rely on tactile end-organs such as Merkel discs, Pacinian corpuscles, Meissner’s corpuscles, and Ruffini endings for environmental exploration, social interaction, tactile discrimination and other tasks in life [11]. Merkel discs, also known as Merkel cell–neurite complexes, are located in high abundance in fingertips of humans, whisker hair follicles of non-human mammals [7,14], and other touch-sensitive spots throughout mammalian body [8,15]. Although discovered 139 years ago [14], only recently have the mechanisms underlying tactile transduction in Merkel discs been uncovered [9,13,19]. Merkel cells have now been established as primary sites of tactile transduction and Piezo2 channels are mechanotransducers in Merkel cells [9,13,19]. Furthermore, it has also been found that Piezo2 channels transduce tactile stimuli into Ca2+ -action potentials in Merkel cells, which drives Aafferent nerve endings to fire slowly adapting impulses and initiate
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∗ Corresponding author. Tel.: +1 513 558 2410. E-mail address: [email protected] (J.G. Gu).
the sense of touch [9]. However, details about Piezo2-mediated tactile transduction in Merkel cells remain to be explored. In heterologous expression system that expressed Piezo1 or Piezo2 channels, membrane displacements evoked whole-cell MA currents (mechanically activated currents) [3]. Piezo1 single channel activity could be elicited by stretching membrane patches using negative pressures when recordings were performed under the cell-attached configuration; the single channel conductance of Piezo1 was measured to be ∼23 and ∼30 pS [3,4]. While wholecell Piezo2 currents have been observed in heterologous expression system, sensory neurons [3], and Merkel cells [9,13,19], single channel properties of Piezo2 channels remain unknown. Single channel properties of ion channels can be directly studied by single channel recording techniques when the channels are accessible by patch-clamp electrodes. When ion channels are not accessible by patch-clamp electrodes, e.g. the channels that are localized at distal domains such as synaptic sites, single channel properties can only be assessed by channel noise fluctuation analyses of whole-cell (macroscopic) currents [10,17]. Structurally, each Merkel cell has a number of finger-like processes that protrude into adjacent epidermal cells [8,14]. It has been thought that these processes might be primary sites of mechanotransduction.
http://dx.doi.org/10.1016/j.neulet.2014.05.055 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: R. Ikeda, J.G. Gu, Piezo2 channel conductance and localization domains in Merkel cells of rat whisker hair follicles, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.055
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Fig. 1. Merkel cell membrane patches under the cell-attached configuration display outward unitary currents with large single channel conductance. (A) Images show Merkel cells in a rat whisker hair follicle. Merkel cells were vital-stained by the fluorescent dye quinacrine and observed under bright field (left panel) and fluorescent field (right). Merkel cells are in green color in the right panel. (B) Sample traces show outward unitary currents recorded from a Merkel cell membrane patch under the cell-attached configuration. The membrane patch was held at trans-membrane potentials from 60 to −60 mV (indicated on the left side of each trace). Channel closing state is indicated by the letter c on the right of each trace. (C) Summary data of I–V relationship of the outward unitary currents (n = 31). (D) Sample traces show outward unitary currents recorded under different negative pressures. The negative pressures were applied using a high-speed pressure-clamp (HSPC) device, and the pressure steps were indicated on the top of recording traces. The Merkel cell was held at 60 mV. (E) Summary data (n = 20) show the channel open time for the outward unitary currents at different negative pressures. Data represent mean ± SEM, NS, not significantly different from the controls recorded without negative pressures (0 mmHg). 58 59 60 61 62 63 64 65 66 67
The processes usually became lost in acutely dissociated Merkel cells, which might account for the lack of mechanical sensitivity in the acutely dissociated Merkel cells [18,20]. Merkel cell processes are too fine to be directly studied for their mechanical sensitivity by electrophysiological methods. Piezo2-mediated mechanical responses in Merkel cells were detected based on the macroscopic MA currents recorded from the cell bodies of Merkel cells. These macroscopic currents may represent the activity of Piezo2 channels that are located on the cell bodies and/or the processes of Merkel cells.
2. Materials and methods Animal care and use conformed to NIH guidelines for care and use of experimental animals. Experimental protocols were approved by the University of Cincinnati Institutional Animal Care and Use Committee. Merkel cells in situ preparations were made as described in our previous study [9]. In brief, Sprague Dawley rats aged 10–18 days were used. Whisker hair follicles were dissected out from whisker pads and the capsule of each follicle was removed. The follicles were then fixed in a recording chamber with
Please cite this article in press as: R. Ikeda, J.G. Gu, Piezo2 channel conductance and localization domains in Merkel cells of rat whisker hair follicles, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.055
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experiments, Cs+ -based internal solution was used and the solution contained (in mM): 140 CsCl, 0.5 CaCl2 , 2 MgCl2 , 5 EGTA, 5 HEPES, 5 Na2 ATP and 0.5 GTP-TRIS salt. Signals were amplified and law-pass filtered at 2 kHz using the Multiclamp 700A amplifier and sampled at 4 kHz using pCLAMP 10 software for most experiments; the recordings for non-stationary fluctuation analysis used law-pass filter of 6 kHz and sampling rate of 50 kHz. To record single channel activity, recordings were made from the cell bodies of Merkel cells under the cell-attached configuration. Membrane patches were held at trans-membrane voltages ranging from −60 mV to +60 mV, which took resting membrane potential of −60 mV into consideration for setting commanding voltages. To deliver mechanical stimulation, steps of negative pressures were applied to the membrane patches through the patch-clamp electrode using a high-speed pressure-clamp device. After the recordings under the cell-attached configuration, the whole-cell configuration was obtained to record whole-cell MA currents.
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a tissue anchor and submerged in a Krebs solution that contained (in mM): 117 NaCl, 3.5 KCl, 2.5 CaCl2 , 1.2 MgCl2 , 1.2 NaH2 PO4 , 25 NaHCO3 and 11 glucose; the solution was saturated with 95% O2 and 5% CO2 , had pH of 7.3 and osmolarity of 325 mOsm, and was maintained at 23 ◦ C. The recording chamber was mounted on the stage of a microscope. The follicles were exposed to 0.05% dispase II plus 0.01% collagenase in the Krebs solution for 8–15 min, and the enzymes were then washed off. Ring sinus cells and the glassy membranes were peeled off using a glass electrode. The follicles were then incubated with 0.3 M quinacrine in the Krebs solution for 15 min to vital-stain Merkel cells. After the staining, the follicles were continually perfused with the Krebs solution. Patch-clamp recordings were made from quinacrine-stained cells described in our previous study [9]. In brief, for most experiments recording electrodes were filled with an internal solution containing (in mM): 135 K-gluconate, 5 KCl, 0.5 CaCl2 , 2 MgCl2 , 5 EGTA, 5 HEPES, 5 Na2 ATP and 0.5 GTP-TRIS salt. In some
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Fig. 2. Applications of negative pressures onto Merkel cell membrane patches did not elicit inward unitary currents. (A) Sample traces show recordings from a Merkel cell membrane patch under the cell-attached configuration. The membrane patch was held at the trans-membrane potential of −60 mV and negative pressures were applied to the membrane patch. Negative pressure steps are indicated on the top of the traces and also on the left side of each trace. (B) Three sample traces each at an expanded scale show noise level before (0 mmHg) and following the applications of negative pressures at 100 and 160 mmHg. (C) Mean amplitude (top panel, n = 46) and variance (bottom panel, n = 46) of the recordings from the membrane patches under different negative pressures up to 190 mmHg. The values at each point were obtained after subtraction of baseline values at 0 mmHg. Data represent mean ± SEM, NS, not significantly different from the value of 0.
Please cite this article in press as: R. Ikeda, J.G. Gu, Piezo2 channel conductance and localization domains in Merkel cells of rat whisker hair follicles, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.055
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Unless otherwise indicated, whole-cell MA currents were recorded at the holding potential of −75 mV. Whole-cell MA currents were elicited by indirect membrane displacement using a fire-polished blunted glass probe [9]. To assess MA channel unitary currents, whole-cell MA currents were evoked by multiple membrane displacements up to 72 times. Non-stationary fluctuation analysis was then applied to the MA currents. The MA channel unitary current (i) was determined by plotting the variance ( 2 ) against mean current (I) and fitting with a parabolic function: 2 = i × I − I2 /N [1,10,17]. The steady-state component of whole-cell MA currents was analyzed for assessing MA channel unitary currents by stationary fluctuation analysis using the equation i = 2 /I [6]. Unless otherwise indicated, membrane voltages mentioned in the texts have been corrected for calculated junction potentials. Data are presented as mean ± SEM. Statistical significance was evaluated using one-way ANOVA with Bonferroni post hoc tests.
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Merkel cells could be specifically vital-stained by the fluorescent dye quinacrine, which allowed us to pre-identify them for patch-clamp recordings (Fig. 1A). Merkel cells in our preparations had elongated cell bodies, similar to the intact Merkel cells described in previous studies [5,14]. We applied patch-clamp recordings on the cell bodies of Merkel cells; the patch-clamp recording electrodes were randomly positioned at different locations on Merkel cell bodies. In most membrane patches, single channel activity could be observed immediately after the formation of the G seal, i.e. the cell-attached configuration (Fig. 1B). Of 44 Merkel cells recorded under the cell-attached configuration, 31 (70.5%) of them showed the single channel activity. The single channel activity was voltagedependent, the unitary currents were outward, and the amplitudes of the unitary currents were larger when membrane patches were
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held at more positive potentials (Fig. 1B and C). The single channel activity was few and often undetectable at holding potentials near or below −40 mV (Fig. 1B and C, n = 31). The single channel conductance was 201 ± 49 pS (n = 31). We determined whether the outward unitary currents in Merkel cells were affected by membrane stretch. The membrane stretch was achieved by applying negative pressures through recording electrodes using a highspeed pressure-clamp device (Fig. 1D). The single channel activity was not significantly different between the recordings without and with negative pressures up to 150 mmHg (n = 20, Fig. 1D and E). We have previously shown that Merkel cell MA currents are mediated by Piezo2 channels and the MA currents are inward at negative holding potentials [9]. We determined whether negative pressures on membrane patches could elicit inward unitary currents (Fig. 2). Under the cell-attached configuration and with Merkel cell membrane patches being held at −60 mV, no inward current could be elicited by negative pressures up to 190 mmHg (Fig. 2B and C, n = 46 cells). Stationary fluctuation analysis was applied to the recording traces before and following the application of negative pressures. No significant change was observed in either baseline currents or signal variances after the application of negative pressures (Fig. 2C). For example, at the negative pressure of 100 mmHg, the mean baseline current amplitude was 0.009 ± 0.061 pA (n = 44) and variance 0.007 ± 0.039 pA2 (n = 44), and both parameters were not significantly different from the value of 0. Thus, there was no appearance of channel activity after the applications of negative pressures to Merkel cell membrane patches (Fig. 2C). While none of the Merkel cells tested responded to negative pressures under the cell-attached configuration, every one of them responded to mechanical stimuli after establishing the whole-cell configuration (Fig. 3). Action potentials could be elicited when depolarizing currents were injected into Merkel cells (Fig. 3A) and large outward whole-cell currents were evoked when depolarizing voltage steps were applied to Merkel cells (Fig. 3B). Under the
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Fig. 3. Membrane displacement stimuli elicited rapidly adapting whole-cell MA currents in Merkel cells. (A)–(C) An example shows that Merkel cell could fire action potentials (A) and had large voltage-activated currents (B) as well as mechanically activated (MA) currents (C) when recorded under the whole-cell configuration. Prior to the whole-cell configuration, the Merkel cell did not respond to negative pressures when the recording was made under the cell-attached configuration. (D) Five consecutive membrane displacement stimuli at 10 Hz (left panel) and 5 Hz (right panel) show the reduction of peak amplitude of MA currents. (E) Sample traces of whole-cell MA currents evoked by five consecutive membrane displacements to show stable peak currents at the frequency of 0.07 Hz. (F) Summary data of the experiments represented in (E) (n = 4). From (C) to (F), cells were held at −75 mV. Data represent mean ± SEM, NS, no significant difference.
Please cite this article in press as: R. Ikeda, J.G. Gu, Piezo2 channel conductance and localization domains in Merkel cells of rat whisker hair follicles, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.055
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voltage-clamp configuration, Merkel cells responded to membrane displacements with inward whole-cell MA currents (Fig. 3C). When membrane displacement stimuli were applied multiple times to a Merkel cell, peak amplitudes became gradually reduced at stimulation frequency of 10 Hz and 5 Hz (Fig. 3D). However, there was no significant change in the current amplitudes at the stimulation frequency of 0.07 Hz (n = 4, Fig. 3E and F). The stable mechanical responses at low stimulation frequency allowed us to apply non-stationary fluctuation analysis to the
whole-cell MA currents in Merkel cells (Fig. 4A). With multiple stimuli by membrane displacements up to 72 times, non-stationary fluctuation analysis for the whole-cell MA currents yielded unitary current amplitudes of 4.05 ± 0.40 pA (n = 4, Fig. 4B) at the holding potential of −75 mV. Accordingly, the single channel conductance was calculated to be 54.0 ± 5.3 pS (n = 4 cells). In addition to the nonstationary phase of the MA currents, Merkel cell MA currents also had small steady-state current component that displayed channel noise (Fig. 4C). The reversal potential of the steady-state currents
Please cite this article in press as: R. Ikeda, J.G. Gu, Piezo2 channel conductance and localization domains in Merkel cells of rat whisker hair follicles, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.055
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was near 0 mV (n = 10, Fig. 4C). The mean amplitude of stead-state current component was enhanced with the increases of membrane displacements (Fig. 4C, n = 25). The signal variance was also increased in a displacement-dependent manner (Fig. 4D, n = 25). With the membrane displacement at 3.5 m, the mean amplitude of the steady-state current was 5.03 ± 0.80 pA (n = 25, Fig. 4D) and the variance 9.94 ± 1.31 pA2 (n = 25, Fig. 4E). We applied stationary fluctuation analysis to the steady-state currents. With membrane displacements from 0.5 to 6 m, calculated unitary currents were similar in their amplitudes and the values of single channel conductance were also not significantly different (Fig. 4F and G). At the membrane displacement of 3.5 m, the unitary current amplitude was calculated to be 2.44 ± 0.28 pA (n = 25) when cells were held at −75 mV. Accordingly, the single channel conductance of the MA channels was calculated to be 32.5 ± 3.8 pS (n = 25).
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are extremely sensitive to mechanical stimulation and Piezo2 MA currents in Merkel cells could be elicited by sub-micrometers of membrane displacements [9,13,19]. Another possibility is that Piezo2 channels were not present in the membrane patches being tested with negative pressures. These channels may be located preferentially at distal domains of Merkel cells such as the finger-like processes that are inaccessible by patch-clamp electrodes. These distal Piezo2 channels could be activated by membrane displacements, which would generate whole-cell MA currents that were detectable under the whole-cell recording configuration. In agreement with this idea, a recent study have suggested that Piezo2 channels are preferentially localized at membrane sub-domains of cultured sensory neurons [16]. Further studies are needed to provide direct evidence about the localization of Piezo2 channels in Merkel cell processes in order to better understand the structure–function of Merkel cells in tactile transduction. Acknowledgements
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In attempting to directly record single channel activity of Piezo2 channels in Merkel cells, we performed recordings under the cellattached configuration and applied negative pressures to stretch membrane patches. Unexpectedly, negative pressures as high as 190 mmHg could not elicit any detectable inward unitary currents in a large number of membrane patches tested. Membrane patches showed outward unitary currents with single channel conductance being ∼200 pS. The single channel properties of the outward unitary currents in Merkel cells are consistent with those of the voltageand calcium-activated potassium channels (BK channels) [12]. The outward unitary currents in Merkel cell membrane patches were also not affected by negative pressures. In sharp contrast, wholecell MA currents could be evoked by membrane displacements in every Merkel cell tested. The whole-cell MA currents showed an enhancement of channel noise. By applying stationary and nonstationary fluctuation analyses to the whole-cell MA currents, we were able to resolve unitary currents flowing through Piezo2 channels in Merkel cells and obtain their single channel conductance. The stationary fluctuation analysis predicted that the single channel conductance of Piezo2 channels in Merkel cells to be ∼30 pS, similar to the single channel conductance of Piezo1 channels [3,4]. The estimated single channel conductance by non-stationary fluctuation analysis was ∼50 pS, similar to the conductance of a low-threshold mechanically sensitive channel identified in sensory neurons [2]. The discrepancy in single channel conductance between stationary and non-stationary phases in our study may suggest the presence of two conduction states for Piezo2 channels in Merkel cells. While under the cell-attached configuration we did not observe any mechanical response to negative pressures up to 190 mmHg, a previous study reported a large baseline current shift when negative pressures of 100–200 mmHg were applied to the membrane patches of acutely dissociated Merkel cells [16]. A baseline current shift could be caused by a change of seal resistance by negative pressures if membrane seal was not very tight. In our experiments, the membrane patches of all cells were tightly sealed onto recording electrodes and seal resistance was over 2 G. Under this condition, we did not observe any baseline current shift by negative pressures in our recordings. There are two possibilities for the lack of inward unitary currents in our experiments using negative pressures to stretch membrane patches. One is that negative pressures are not adequate stimuli to activate Piezo2 channels even though these channels were present in the Merkel cell membrane patches in our experiments. However, this possibility is discounted by the fact that Piezo1, which is structurally similar to Piezo2, could be activated by a negative pressure as small as 10 mmHg [3]. Furthermore, Piezo2 channels
We thank Dr. D. Coyle for his comments on an earlier version of this manuscript. This work was supported by NIH grants DE018661 Q3 and DE023090 to J.G.G. References [1] O. Alvarez, C. Gonzalez, R. Latorre, Counting channels: a tutorial guide on ion channel fluctuation analysis, Adv. Physiol. Educ. 26 (2002) 327–341. [2] H. Cho, J. Shin, C.Y. Shin, S.Y. Lee, U. Oh, Mechanosensitive ion channels in cultured sensory neurons of neonatal rats, J. Neurosci. 22 (2002) 1238–1247. [3] B. Coste, J. Mathur, M. Schmidt, T.J. Earley, S. Ranade, M.J. Petrus, A.E. Dubin, A. Patapoutian, Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels, Science 330 (2010) 55–60. [4] B. Coste, B. Xiao, J.S. Santos, R. Syeda, J. Grandl, K.S. Spencer, S.E. Kim, M. Schmidt, J. Mathur, A.E. Dubin, M. Montal, A. Patapoutian, Piezo proteins are pore-forming subunits of mechanically activated channels, Nature 483 (2012) 176–181. [5] J. Fukuda, H. Ishimine, Y. Masaki, Long-term staining of live Merkel cells with FM dyes, Cell Tissue Res. 311 (2003) 325–332. [6] P.T.A. Gray, Analysis of whole cell currents to estimate the kinetics and amplitude of underlying unitary events: relaxation and “noise” analysis, in: D. Ogden (Ed.), Microelectrode Techniques, The Company of Biologists, Cambridge, 1987. [7] K. Hashimoto, The ultrastructure of the skin of human embryos. X. Merkel tactile cells in the finger and nail, J. Anat. 111 (1972) 99–120. [8] A. Iggo, A.R. Muir, The structure and function of a slowly adapting touch corpuscle in hairy skin, J. Physiol. 200 (1969) 763–796. [9] R. Ikeda, M. Cha, J. Ling, Z. Jia, D. Coyle, J.G. Gu, Merkel cells transduce and encode tactile stimuli to drive abeta-afferent impulses, Cell 157 (2014) 664–675. [10] A.C. Jackson, A.D. Milstein, D. Soto, M. Farrant, S.G. Cull-Candy, R.A. Nicoll, Probing TARP modulation of AMPA receptor conductance with polyamine toxins, J. Neurosci. 31 (2011) 7511–7520. [11] K.O. Johnson, The roles and functions of cutaneous mechanoreceptors, Curr. Opin. Neurobiol. 11 (2001) 455–461. [12] U.S. Lee, J. Cui, BK channel activation: structural and functional insights, Trends Neurosci. 33 (2010) 415–423. [13] S. Maksimovic, M. Nakatani, Y. Baba, A.M. Nelson, K.L. Marshall, S.A. Wellnitz, P. Firozi, S.H. Woo, S. Ranade, A. Patapoutian, E.A. Lumpkin, Epidermal Merkel cells are mechanosensory cells that tune mammalian touch receptors, Nature Q4 (2014). [14] F. Merkel, Tastzellen and Tastkoerperchen bei den Hausthieren und beim Menschen, Arch. Mikrosc. Anat. 11 (1875) 636–652. [15] B.L. Munger, The intraepidermal innervation of the snout skin of the opossum. A light and electron microscope study, with observations on the nature of Merkel’s Tastzellen, J. Cell Biol. 26 (1965) 79–97. [16] K. Poole, R. Herget, L. Lapatsina, H.D. Ngo, G.R. Lewin, Tuning Piezo ion channels to detect molecular-scale movements relevant for fine touch, Nat. Commun. 5 (2014) 3520. [17] F.J. Sigworth, The variance of sodium current fluctuations at the node of Ranvier, J. Physiol. 307 (1980) 97–129. [18] Y. Tazaki, M. Tazaki, T. Inoue, M. Shimono, Scanning and transmission electron microscopic observation of changes in cylindrical cytoplasmic processes of isolated single merkel cell, Bull Tokyo Dent. Coll. 52 (2011) 69–76. [19] S.H. Woo, S. Ranade, A.D. Weyer, A.E. Dubin, Y. Baba, Z. Qiu, M. Petrus, T. Miyamoto, K. Reddy, E.A. Lumpkin, C.L. Stucky, A. Patapoutian, Piezo2 is required for Merkel-cell mechanotransduction, Nature (2014). [20] Y. Yamashita, N. Akaike, M. Wakamori, I. Ikeda, H. Ogawa, Voltage-dependent currents in isolated single Merkel cells of rats, J. Physiol. 450 (1992) 143–162.
Please cite this article in press as: R. Ikeda, J.G. Gu, Piezo2 channel conductance and localization domains in Merkel cells of rat whisker hair follicles, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.055
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Plenary Article
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Piezo2 channel conductance and localization domains in Merkel cells of rat whisker hair follicles
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Ryo Ikeda a,b , Jianguo G. Gu a,∗ a
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Department of Anesthesiology, The University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0531, USA Department of Orthopaedic Surgery, Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan
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Keywords: Merkel cell Mechanotransduction Mechanoreceptor Piezo2 Whisker hair follicle
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1. Introduction
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We have recently shown that Merkel cells transduce tactile stimuli via Piezo2 channels to initiate the sense of touch. Here we performed patch-clamp recordings to assess single channel activity on the membranes of Merkel cells in whisker hair follicles. Under the cell-attached configuration, most Merkel cell membrane patches showed large outward unitary currents with single channel conductance being ∼200 pS. The outward unitary currents were not affected by negative pressures up to 150 mmHg when applied to the membrane patches. The application of negative pressures up to 190 mmHg also could not directly elicit any inward unitary current in the membrane patches. However, after establishing the whole-cell configuration, mechanically activated currents (MA) that resembled Piezo2 currents could be elicited by membrane displacements in every Merkel cell tested. While the MA current decayed rapidly, a small steady-state current component with significant channel noise could be observed. Applications of stationary and non-stationary fluctuation analyses to the MA currents yielded single channel conductance of 32.5 ± 3.8 and 54.0 ± 5.3 pS, respectively. The lack of mechanical responses under the cell-attached configuration and the existence of Piezo2 MA currents under the whole-cell configuration raised a possibility that Piezo2 channels are preferentially located on Merkel cell processes, the membrane domains inaccessible by recording electrodes. © 2014 Published by Elsevier Ireland Ltd.
Mammals rely on tactile end-organs such as Merkel discs, Pacinian corpuscles, Meissner’s corpuscles, and Ruffini endings for environmental exploration, social interaction, tactile discrimination and other tasks in life [11]. Merkel discs, also known as Merkel cell–neurite complexes, are located in high abundance in fingertips of humans, whisker hair follicles of non-human mammals [7,14], and other touch-sensitive spots throughout mammalian body [8,15]. Although discovered 139 years ago [14], only recently have the mechanisms underlying tactile transduction in Merkel discs been uncovered [9,13,19]. Merkel cells have now been established as primary sites of tactile transduction and Piezo2 channels are mechanotransducers in Merkel cells [9,13,19]. Furthermore, it has also been found that Piezo2 channels transduce tactile stimuli into Ca2+ -action potentials in Merkel cells, which drives Aafferent nerve endings to fire slowly adapting impulses and initiate
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∗ Corresponding author. Tel.: +1 513 558 2410. E-mail address: [email protected] (J.G. Gu).
the sense of touch [9]. However, details about Piezo2-mediated tactile transduction in Merkel cells remain to be explored. In heterologous expression system that expressed Piezo1 or Piezo2 channels, membrane displacements evoked whole-cell MA currents (mechanically activated currents) [3]. Piezo1 single channel activity could be elicited by stretching membrane patches using negative pressures when recordings were performed under the cell-attached configuration; the single channel conductance of Piezo1 was measured to be ∼23 and ∼30 pS [3,4]. While wholecell Piezo2 currents have been observed in heterologous expression system, sensory neurons [3], and Merkel cells [9,13,19], single channel properties of Piezo2 channels remain unknown. Single channel properties of ion channels can be directly studied by single channel recording techniques when the channels are accessible by patch-clamp electrodes. When ion channels are not accessible by patch-clamp electrodes, e.g. the channels that are localized at distal domains such as synaptic sites, single channel properties can only be assessed by channel noise fluctuation analyses of whole-cell (macroscopic) currents [10,17]. Structurally, each Merkel cell has a number of finger-like processes that protrude into adjacent epidermal cells [8,14]. It has been thought that these processes might be primary sites of mechanotransduction.
http://dx.doi.org/10.1016/j.neulet.2014.05.055 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: R. Ikeda, J.G. Gu, Piezo2 channel conductance and localization domains in Merkel cells of rat whisker hair follicles, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.055
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Fig. 1. Merkel cell membrane patches under the cell-attached configuration display outward unitary currents with large single channel conductance. (A) Images show Merkel cells in a rat whisker hair follicle. Merkel cells were vital-stained by the fluorescent dye quinacrine and observed under bright field (left panel) and fluorescent field (right). Merkel cells are in green color in the right panel. (B) Sample traces show outward unitary currents recorded from a Merkel cell membrane patch under the cell-attached configuration. The membrane patch was held at trans-membrane potentials from 60 to −60 mV (indicated on the left side of each trace). Channel closing state is indicated by the letter c on the right of each trace. (C) Summary data of I–V relationship of the outward unitary currents (n = 31). (D) Sample traces show outward unitary currents recorded under different negative pressures. The negative pressures were applied using a high-speed pressure-clamp (HSPC) device, and the pressure steps were indicated on the top of recording traces. The Merkel cell was held at 60 mV. (E) Summary data (n = 20) show the channel open time for the outward unitary currents at different negative pressures. Data represent mean ± SEM, NS, not significantly different from the controls recorded without negative pressures (0 mmHg). 58 59 60 61 62 63 64 65 66 67
The processes usually became lost in acutely dissociated Merkel cells, which might account for the lack of mechanical sensitivity in the acutely dissociated Merkel cells [18,20]. Merkel cell processes are too fine to be directly studied for their mechanical sensitivity by electrophysiological methods. Piezo2-mediated mechanical responses in Merkel cells were detected based on the macroscopic MA currents recorded from the cell bodies of Merkel cells. These macroscopic currents may represent the activity of Piezo2 channels that are located on the cell bodies and/or the processes of Merkel cells.
2. Materials and methods Animal care and use conformed to NIH guidelines for care and use of experimental animals. Experimental protocols were approved by the University of Cincinnati Institutional Animal Care and Use Committee. Merkel cells in situ preparations were made as described in our previous study [9]. In brief, Sprague Dawley rats aged 10–18 days were used. Whisker hair follicles were dissected out from whisker pads and the capsule of each follicle was removed. The follicles were then fixed in a recording chamber with
Please cite this article in press as: R. Ikeda, J.G. Gu, Piezo2 channel conductance and localization domains in Merkel cells of rat whisker hair follicles, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.055
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experiments, Cs+ -based internal solution was used and the solution contained (in mM): 140 CsCl, 0.5 CaCl2 , 2 MgCl2 , 5 EGTA, 5 HEPES, 5 Na2 ATP and 0.5 GTP-TRIS salt. Signals were amplified and law-pass filtered at 2 kHz using the Multiclamp 700A amplifier and sampled at 4 kHz using pCLAMP 10 software for most experiments; the recordings for non-stationary fluctuation analysis used law-pass filter of 6 kHz and sampling rate of 50 kHz. To record single channel activity, recordings were made from the cell bodies of Merkel cells under the cell-attached configuration. Membrane patches were held at trans-membrane voltages ranging from −60 mV to +60 mV, which took resting membrane potential of −60 mV into consideration for setting commanding voltages. To deliver mechanical stimulation, steps of negative pressures were applied to the membrane patches through the patch-clamp electrode using a high-speed pressure-clamp device. After the recordings under the cell-attached configuration, the whole-cell configuration was obtained to record whole-cell MA currents.
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a tissue anchor and submerged in a Krebs solution that contained (in mM): 117 NaCl, 3.5 KCl, 2.5 CaCl2 , 1.2 MgCl2 , 1.2 NaH2 PO4 , 25 NaHCO3 and 11 glucose; the solution was saturated with 95% O2 and 5% CO2 , had pH of 7.3 and osmolarity of 325 mOsm, and was maintained at 23 ◦ C. The recording chamber was mounted on the stage of a microscope. The follicles were exposed to 0.05% dispase II plus 0.01% collagenase in the Krebs solution for 8–15 min, and the enzymes were then washed off. Ring sinus cells and the glassy membranes were peeled off using a glass electrode. The follicles were then incubated with 0.3 M quinacrine in the Krebs solution for 15 min to vital-stain Merkel cells. After the staining, the follicles were continually perfused with the Krebs solution. Patch-clamp recordings were made from quinacrine-stained cells described in our previous study [9]. In brief, for most experiments recording electrodes were filled with an internal solution containing (in mM): 135 K-gluconate, 5 KCl, 0.5 CaCl2 , 2 MgCl2 , 5 EGTA, 5 HEPES, 5 Na2 ATP and 0.5 GTP-TRIS salt. In some
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Fig. 2. Applications of negative pressures onto Merkel cell membrane patches did not elicit inward unitary currents. (A) Sample traces show recordings from a Merkel cell membrane patch under the cell-attached configuration. The membrane patch was held at the trans-membrane potential of −60 mV and negative pressures were applied to the membrane patch. Negative pressure steps are indicated on the top of the traces and also on the left side of each trace. (B) Three sample traces each at an expanded scale show noise level before (0 mmHg) and following the applications of negative pressures at 100 and 160 mmHg. (C) Mean amplitude (top panel, n = 46) and variance (bottom panel, n = 46) of the recordings from the membrane patches under different negative pressures up to 190 mmHg. The values at each point were obtained after subtraction of baseline values at 0 mmHg. Data represent mean ± SEM, NS, not significantly different from the value of 0.
Please cite this article in press as: R. Ikeda, J.G. Gu, Piezo2 channel conductance and localization domains in Merkel cells of rat whisker hair follicles, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.055
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Unless otherwise indicated, whole-cell MA currents were recorded at the holding potential of −75 mV. Whole-cell MA currents were elicited by indirect membrane displacement using a fire-polished blunted glass probe [9]. To assess MA channel unitary currents, whole-cell MA currents were evoked by multiple membrane displacements up to 72 times. Non-stationary fluctuation analysis was then applied to the MA currents. The MA channel unitary current (i) was determined by plotting the variance ( 2 ) against mean current (I) and fitting with a parabolic function: 2 = i × I − I2 /N [1,10,17]. The steady-state component of whole-cell MA currents was analyzed for assessing MA channel unitary currents by stationary fluctuation analysis using the equation i = 2 /I [6]. Unless otherwise indicated, membrane voltages mentioned in the texts have been corrected for calculated junction potentials. Data are presented as mean ± SEM. Statistical significance was evaluated using one-way ANOVA with Bonferroni post hoc tests.
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3. Results
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Merkel cells could be specifically vital-stained by the fluorescent dye quinacrine, which allowed us to pre-identify them for patch-clamp recordings (Fig. 1A). Merkel cells in our preparations had elongated cell bodies, similar to the intact Merkel cells described in previous studies [5,14]. We applied patch-clamp recordings on the cell bodies of Merkel cells; the patch-clamp recording electrodes were randomly positioned at different locations on Merkel cell bodies. In most membrane patches, single channel activity could be observed immediately after the formation of the G seal, i.e. the cell-attached configuration (Fig. 1B). Of 44 Merkel cells recorded under the cell-attached configuration, 31 (70.5%) of them showed the single channel activity. The single channel activity was voltagedependent, the unitary currents were outward, and the amplitudes of the unitary currents were larger when membrane patches were
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held at more positive potentials (Fig. 1B and C). The single channel activity was few and often undetectable at holding potentials near or below −40 mV (Fig. 1B and C, n = 31). The single channel conductance was 201 ± 49 pS (n = 31). We determined whether the outward unitary currents in Merkel cells were affected by membrane stretch. The membrane stretch was achieved by applying negative pressures through recording electrodes using a highspeed pressure-clamp device (Fig. 1D). The single channel activity was not significantly different between the recordings without and with negative pressures up to 150 mmHg (n = 20, Fig. 1D and E). We have previously shown that Merkel cell MA currents are mediated by Piezo2 channels and the MA currents are inward at negative holding potentials [9]. We determined whether negative pressures on membrane patches could elicit inward unitary currents (Fig. 2). Under the cell-attached configuration and with Merkel cell membrane patches being held at −60 mV, no inward current could be elicited by negative pressures up to 190 mmHg (Fig. 2B and C, n = 46 cells). Stationary fluctuation analysis was applied to the recording traces before and following the application of negative pressures. No significant change was observed in either baseline currents or signal variances after the application of negative pressures (Fig. 2C). For example, at the negative pressure of 100 mmHg, the mean baseline current amplitude was 0.009 ± 0.061 pA (n = 44) and variance 0.007 ± 0.039 pA2 (n = 44), and both parameters were not significantly different from the value of 0. Thus, there was no appearance of channel activity after the applications of negative pressures to Merkel cell membrane patches (Fig. 2C). While none of the Merkel cells tested responded to negative pressures under the cell-attached configuration, every one of them responded to mechanical stimuli after establishing the whole-cell configuration (Fig. 3). Action potentials could be elicited when depolarizing currents were injected into Merkel cells (Fig. 3A) and large outward whole-cell currents were evoked when depolarizing voltage steps were applied to Merkel cells (Fig. 3B). Under the
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Fig. 3. Membrane displacement stimuli elicited rapidly adapting whole-cell MA currents in Merkel cells. (A)–(C) An example shows that Merkel cell could fire action potentials (A) and had large voltage-activated currents (B) as well as mechanically activated (MA) currents (C) when recorded under the whole-cell configuration. Prior to the whole-cell configuration, the Merkel cell did not respond to negative pressures when the recording was made under the cell-attached configuration. (D) Five consecutive membrane displacement stimuli at 10 Hz (left panel) and 5 Hz (right panel) show the reduction of peak amplitude of MA currents. (E) Sample traces of whole-cell MA currents evoked by five consecutive membrane displacements to show stable peak currents at the frequency of 0.07 Hz. (F) Summary data of the experiments represented in (E) (n = 4). From (C) to (F), cells were held at −75 mV. Data represent mean ± SEM, NS, no significant difference.
Please cite this article in press as: R. Ikeda, J.G. Gu, Piezo2 channel conductance and localization domains in Merkel cells of rat whisker hair follicles, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.055
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Fig. 4. Stationary and non-stationary analyses of whole-cell MA currents in Merkel cells. (A) Sample trace (light gray color) shows a whole-cell MA current elicited by a membrane displacement at 4 m. The dark black line represents the average of 72 traces that were generated by repeated membrane displacement stimuli at 0.07 Hz. (B) Non-stationary fluctuation analyses by plotting the variances at different current amplitudes. Similar results were obtained from 3 other Merkel cells. The mean unitary currents were 4.05 ± 0.40 pA (n = 4 Merkel cells). (C) Left and middle panels show an example of steady-state current component of a whole-cell MA current recorded from a Merkel cell. Channel noise can be seen during steady-state current phase at an expanded scale (middle panel). Right panel shows I–V blot of the steady-state currents (n = 10). (D)–(G) Stationary fluctuation analyses of the steady-state currents (n = 25) show mean amplitude of the steady-state currents (D) and their variance (E) as well as the calculated unitary currents (F) and single channel conductance (G). Data represent mean ± SEM, NS, no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001.
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voltage-clamp configuration, Merkel cells responded to membrane displacements with inward whole-cell MA currents (Fig. 3C). When membrane displacement stimuli were applied multiple times to a Merkel cell, peak amplitudes became gradually reduced at stimulation frequency of 10 Hz and 5 Hz (Fig. 3D). However, there was no significant change in the current amplitudes at the stimulation frequency of 0.07 Hz (n = 4, Fig. 3E and F). The stable mechanical responses at low stimulation frequency allowed us to apply non-stationary fluctuation analysis to the
whole-cell MA currents in Merkel cells (Fig. 4A). With multiple stimuli by membrane displacements up to 72 times, non-stationary fluctuation analysis for the whole-cell MA currents yielded unitary current amplitudes of 4.05 ± 0.40 pA (n = 4, Fig. 4B) at the holding potential of −75 mV. Accordingly, the single channel conductance was calculated to be 54.0 ± 5.3 pS (n = 4 cells). In addition to the nonstationary phase of the MA currents, Merkel cell MA currents also had small steady-state current component that displayed channel noise (Fig. 4C). The reversal potential of the steady-state currents
Please cite this article in press as: R. Ikeda, J.G. Gu, Piezo2 channel conductance and localization domains in Merkel cells of rat whisker hair follicles, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.055
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was near 0 mV (n = 10, Fig. 4C). The mean amplitude of stead-state current component was enhanced with the increases of membrane displacements (Fig. 4C, n = 25). The signal variance was also increased in a displacement-dependent manner (Fig. 4D, n = 25). With the membrane displacement at 3.5 m, the mean amplitude of the steady-state current was 5.03 ± 0.80 pA (n = 25, Fig. 4D) and the variance 9.94 ± 1.31 pA2 (n = 25, Fig. 4E). We applied stationary fluctuation analysis to the steady-state currents. With membrane displacements from 0.5 to 6 m, calculated unitary currents were similar in their amplitudes and the values of single channel conductance were also not significantly different (Fig. 4F and G). At the membrane displacement of 3.5 m, the unitary current amplitude was calculated to be 2.44 ± 0.28 pA (n = 25) when cells were held at −75 mV. Accordingly, the single channel conductance of the MA channels was calculated to be 32.5 ± 3.8 pS (n = 25).
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are extremely sensitive to mechanical stimulation and Piezo2 MA currents in Merkel cells could be elicited by sub-micrometers of membrane displacements [9,13,19]. Another possibility is that Piezo2 channels were not present in the membrane patches being tested with negative pressures. These channels may be located preferentially at distal domains of Merkel cells such as the finger-like processes that are inaccessible by patch-clamp electrodes. These distal Piezo2 channels could be activated by membrane displacements, which would generate whole-cell MA currents that were detectable under the whole-cell recording configuration. In agreement with this idea, a recent study have suggested that Piezo2 channels are preferentially localized at membrane sub-domains of cultured sensory neurons [16]. Further studies are needed to provide direct evidence about the localization of Piezo2 channels in Merkel cell processes in order to better understand the structure–function of Merkel cells in tactile transduction. Acknowledgements
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In attempting to directly record single channel activity of Piezo2 channels in Merkel cells, we performed recordings under the cellattached configuration and applied negative pressures to stretch membrane patches. Unexpectedly, negative pressures as high as 190 mmHg could not elicit any detectable inward unitary currents in a large number of membrane patches tested. Membrane patches showed outward unitary currents with single channel conductance being ∼200 pS. The single channel properties of the outward unitary currents in Merkel cells are consistent with those of the voltageand calcium-activated potassium channels (BK channels) [12]. The outward unitary currents in Merkel cell membrane patches were also not affected by negative pressures. In sharp contrast, wholecell MA currents could be evoked by membrane displacements in every Merkel cell tested. The whole-cell MA currents showed an enhancement of channel noise. By applying stationary and nonstationary fluctuation analyses to the whole-cell MA currents, we were able to resolve unitary currents flowing through Piezo2 channels in Merkel cells and obtain their single channel conductance. The stationary fluctuation analysis predicted that the single channel conductance of Piezo2 channels in Merkel cells to be ∼30 pS, similar to the single channel conductance of Piezo1 channels [3,4]. The estimated single channel conductance by non-stationary fluctuation analysis was ∼50 pS, similar to the conductance of a low-threshold mechanically sensitive channel identified in sensory neurons [2]. The discrepancy in single channel conductance between stationary and non-stationary phases in our study may suggest the presence of two conduction states for Piezo2 channels in Merkel cells. While under the cell-attached configuration we did not observe any mechanical response to negative pressures up to 190 mmHg, a previous study reported a large baseline current shift when negative pressures of 100–200 mmHg were applied to the membrane patches of acutely dissociated Merkel cells [16]. A baseline current shift could be caused by a change of seal resistance by negative pressures if membrane seal was not very tight. In our experiments, the membrane patches of all cells were tightly sealed onto recording electrodes and seal resistance was over 2 G. Under this condition, we did not observe any baseline current shift by negative pressures in our recordings. There are two possibilities for the lack of inward unitary currents in our experiments using negative pressures to stretch membrane patches. One is that negative pressures are not adequate stimuli to activate Piezo2 channels even though these channels were present in the Merkel cell membrane patches in our experiments. However, this possibility is discounted by the fact that Piezo1, which is structurally similar to Piezo2, could be activated by a negative pressure as small as 10 mmHg [3]. Furthermore, Piezo2 channels
We thank Dr. D. Coyle for his comments on an earlier version of this manuscript. This work was supported by NIH grants DE018661 Q3 and DE023090 to J.G.G. References [1] O. Alvarez, C. Gonzalez, R. Latorre, Counting channels: a tutorial guide on ion channel fluctuation analysis, Adv. Physiol. Educ. 26 (2002) 327–341. [2] H. Cho, J. Shin, C.Y. Shin, S.Y. Lee, U. Oh, Mechanosensitive ion channels in cultured sensory neurons of neonatal rats, J. Neurosci. 22 (2002) 1238–1247. [3] B. Coste, J. Mathur, M. Schmidt, T.J. Earley, S. Ranade, M.J. Petrus, A.E. Dubin, A. Patapoutian, Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels, Science 330 (2010) 55–60. [4] B. Coste, B. Xiao, J.S. Santos, R. Syeda, J. Grandl, K.S. Spencer, S.E. Kim, M. Schmidt, J. Mathur, A.E. Dubin, M. Montal, A. Patapoutian, Piezo proteins are pore-forming subunits of mechanically activated channels, Nature 483 (2012) 176–181. [5] J. Fukuda, H. Ishimine, Y. Masaki, Long-term staining of live Merkel cells with FM dyes, Cell Tissue Res. 311 (2003) 325–332. [6] P.T.A. Gray, Analysis of whole cell currents to estimate the kinetics and amplitude of underlying unitary events: relaxation and “noise” analysis, in: D. Ogden (Ed.), Microelectrode Techniques, The Company of Biologists, Cambridge, 1987. [7] K. Hashimoto, The ultrastructure of the skin of human embryos. X. Merkel tactile cells in the finger and nail, J. Anat. 111 (1972) 99–120. [8] A. Iggo, A.R. Muir, The structure and function of a slowly adapting touch corpuscle in hairy skin, J. Physiol. 200 (1969) 763–796. [9] R. Ikeda, M. Cha, J. Ling, Z. Jia, D. Coyle, J.G. Gu, Merkel cells transduce and encode tactile stimuli to drive abeta-afferent impulses, Cell 157 (2014) 664–675. [10] A.C. Jackson, A.D. Milstein, D. Soto, M. Farrant, S.G. Cull-Candy, R.A. Nicoll, Probing TARP modulation of AMPA receptor conductance with polyamine toxins, J. Neurosci. 31 (2011) 7511–7520. [11] K.O. Johnson, The roles and functions of cutaneous mechanoreceptors, Curr. Opin. Neurobiol. 11 (2001) 455–461. [12] U.S. Lee, J. Cui, BK channel activation: structural and functional insights, Trends Neurosci. 33 (2010) 415–423. [13] S. Maksimovic, M. Nakatani, Y. Baba, A.M. Nelson, K.L. Marshall, S.A. Wellnitz, P. Firozi, S.H. Woo, S. Ranade, A. Patapoutian, E.A. Lumpkin, Epidermal Merkel cells are mechanosensory cells that tune mammalian touch receptors, Nature Q4 (2014). [14] F. Merkel, Tastzellen and Tastkoerperchen bei den Hausthieren und beim Menschen, Arch. Mikrosc. Anat. 11 (1875) 636–652. [15] B.L. Munger, The intraepidermal innervation of the snout skin of the opossum. A light and electron microscope study, with observations on the nature of Merkel’s Tastzellen, J. Cell Biol. 26 (1965) 79–97. [16] K. Poole, R. Herget, L. Lapatsina, H.D. Ngo, G.R. Lewin, Tuning Piezo ion channels to detect molecular-scale movements relevant for fine touch, Nat. Commun. 5 (2014) 3520. [17] F.J. Sigworth, The variance of sodium current fluctuations at the node of Ranvier, J. Physiol. 307 (1980) 97–129. [18] Y. Tazaki, M. Tazaki, T. Inoue, M. Shimono, Scanning and transmission electron microscopic observation of changes in cylindrical cytoplasmic processes of isolated single merkel cell, Bull Tokyo Dent. Coll. 52 (2011) 69–76. [19] S.H. Woo, S. Ranade, A.D. Weyer, A.E. Dubin, Y. Baba, Z. Qiu, M. Petrus, T. Miyamoto, K. Reddy, E.A. Lumpkin, C.L. Stucky, A. Patapoutian, Piezo2 is required for Merkel-cell mechanotransduction, Nature (2014). [20] Y. Yamashita, N. Akaike, M. Wakamori, I. Ikeda, H. Ogawa, Voltage-dependent currents in isolated single Merkel cells of rats, J. Physiol. 450 (1992) 143–162.
Please cite this article in press as: R. Ikeda, J.G. Gu, Piezo2 channel conductance and localization domains in Merkel cells of rat whisker hair follicles, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.055
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