Voltage‐dependent currents in isolated single Merkel cells of rats.

Abstract

SUMMARY1. Merkel cells were dissociated enzymatically from the footpad epidermis of 10-to 20-day-old rats pretreated with fluorescent dye, quinacrine, for purposes of staining. The fluorescent Merkel cells had an elongated or elliptic shape in situ, yet the dissociated ones were round (7-12 #m in diameter).2. Electrical recordings were performed in the whole-cell configuration using a conventional patch-clamp technique. The mean resting membrane potential of fluorescent Merkel cells was -54 0 mV, the value being greater than the -2641 mV of non-fluorescent epidermal cells. No voltage-dependent channel was observed in non-fluorescent cells.3. The Merkel cells had no Na+ spike in an external standard solution, but tetrodotoxin-resistant long-lasting action potentials were evoked by depolarization with injection of constant currents in an external solution containing Ba2+.4. In Merkel cells under voltage clamp, depolarizing step pulses (800 ms) from a holding potential (VH) of -80 mV elicited predominantly outward K+ currents composed of transient and sustained components: the former was selectively inhibited by 4-aminopyridine (4-AP), while the latter was inhibited by both tetraethylammonium (TEA) and quinacrine. Quinacrine was more effective and selective than TEA in blocking the sustained K+ current but had no effect on the current at the low concentration (10-7or 3 x 10`6 M) used for staining the Merkel cells.5. The sustained outward K+ current (IKD) was activated at potentials more positive than -20 or -10 mV at a VH of -50 mV, at which potential the transient outward K+ channel was completely inactivated. The potential for half-inactivation in the steady-state inactivation curve for IKD was -33 mV.6. The transient outward K+ current (IA) was activated at potentials more positive than -50 mV at a VH of -80 mV. The potential for half-inactivation in the steady-state inactivation curve for IA was -64 mV.7. When the outward K+ currents were blocked by adding both TEA and 4-AP, only a sustained inward Ca2+ current was observed. In an external solution containing 10 mM-Ca2+, ICa was evoked by potentials more positive than -20 mV at a VH of -80 mV, and the maximum inward current appeared around +10 mV. 8. In external solution containing 25 mM-Ba2 , the potential for half-inactivation of the steady-state inactivation curve for IBa was -34 mV.9. The selectivity of the Ca2+ channel for divalent cations was in the order Ba2+ > Sr2+ > Ca2+.10. Organic and inorganic Ca2+ antagonists blocked IBa in a concentrationdependent manner, the potency of inhibition being in the order w-conotoxin > flunarizine = nicardipine > diltiazem > verapamil, and La3+ > Cd2+ > Co2+.11. It is concluded that Merkel cells have at least three distinct voltage-gated currents: a delayed K+ current (IKD), a transient A-current (IA) and a high-threshold (L-type) Ca2+ current (ICa). The electrical and pharmacological properties of these voltage-dependent channels are essentially similar to those of other excitable cells.

Molecular profiling reveals synaptic release machinery in Merkel cells

“…In this study, we used these methods to ascertain which voltagegated Ca 2ϩ channels are active in murine Merkel cells. Our results extend a previous report of Ca 2ϩ currents in Merkel cells (49). We found that almost all of the depolarization-induced Ca 2ϩ influx in Merkel cells is through two types of channels.…”

Epidermal progenitors give rise to Merkel cells during embryonic development and adult homeostasis

“…MCs express intermediate filaments of primitive and simple epithelia such as keratin 8 (K8), K18, or K20 but also express neuropeptides and many components of the presynaptic machinery such as synaptotagmin or Rab3c and transcription factors involved in neuronal cell fate determination (Haeberle et al, 2004). An electrophysiological study demonstrated that MCs are excitable cells (Yamashita et al, 1992) that express voltagegated channels, inducing calcium influx in response to depolarization (Haeberle et al, 2004). Selective destruction of MCs by photoablation (Ikeda et al, 1994) or their loss in mice genetically deficient for MCs (Maricich et al, 2009) abolishes responses of slowly adapting type I mechanoreceptor units, which is consistent with the requirement of MCs to mediate slow adapting mechanotransduction in the skin.…”

Scanning and Transmission Electron Microscopic Observation of Changes in Cylindrical Cytoplasmic Processes of Isolated Single Merkel Cell

“…The calcium flowing into the cell subsequently produces a signal, causing a release of transmitters within the dense-cored granules, resulting in generation of action potential on a connected nerve fiber. There is some evidence to support this hypothesis: the presence of voltage dependent Ca ‫ם2‬ channels 4,29) ; chemical dependent channels 4) ; and stretch activated ion channels 23,25) in Merkel cells. This suggests, therefore, that Merkel cells are not only mechanoreceptors, but also mechano-electric transducers.…”

“…To obtain mechanical stimulation at a high concentration of KCl (KCl, 100 mM; NaCl, 4.1 mM; CaCl, 2.5 mM; MgCl 2 , 0.5 mM; NaHCO 3 , 12 mM; C 6 H 12 O 6 , 11 mM; NaH 2 PO 4 • 2H 2 O, 1.2 mM), single Merkel cells were subjected to depolarized simulation with normal Krebs solution using a Y-tube ( Fig. 1), which allows exchange of whole external solution within 10-20 msec 1,20,25,29) . The presence of voltagedependent Ca ‫ם2‬ channels was confirmed in almost all the fluorescent cells.…”

“…To facilitate separation of epithelium from lamina propria, excised mucosa was treated with Krebs solution containing DL-Dithiothreitol (2 mg/ml, Sigma Chemical Co.) at 31°C for 10 min. The epithelium was then treated with a Ca 1,4,20,25,26,29,30) . Positions of isolated single Merkel cells were marked on the tissue culture dish.…”