Korean J Physiol Pharmacol.  2010 Dec;14(6):419-425. 10.4196/kjpp.2010.14.6.419.

Functional Expression of TRPV4 Cation Channels in Human Mast Cell Line (HMC-1)

• Kim KS
• Shin DH
• Nam JH
• Park KS
• Zhang YH
• Kim WK
• Kim SJ

• KMID: 2071715
• DOI: http://doi.org/10.4196/kjpp.2010.14.6.419

Abstract

Mast cells are activated by specific allergens and also by various nonspecific stimuli, which might induce physical urticaria. This study investigated the functional expression of temperature sensitive transient receptor potential vanilloid (TRPV) subfamily in the human mast cell line (HMC-1) using whole-cell patch clamp techniques. The temperature of perfusate was raised from room temperature (RT, 23~25degrees C to a moderately high temperature (MHT, 37~39degrees C to activate TRPV3/4, a high temperature (HT, 44~46degrees C to activate TRPV1, or a very high temperature (VHT, 53~55degrees C to activate TRPV2. The membrane conductance of HMC-1 was increased by MHT and HT in about 50% (21 of 40) of the tested cells, and the I/V curves showed weak outward rectification. VHT-induced current was 10-fold larger than those induced by MHT and HT. The application of the TRPV4 activator 4alpha-phorbol 12,13-didecanoate (4alphaPDD, 1microM) induced weakly outward rectifying currents similar to those induced by MHT. However, the TRPV3 agonist camphor or TRPV1 agonist capsaicin had no effect. RT-PCR analysis of HMC-1 demonstrated the expression of TRPV4 as well as potent expression of TRPV2. The [Ca2+]c of HMC-1 cells was also increased by MHT or by 4alphaPDD. In summary, our present study indicates that HMC-1 cells express Ca2+-permeable TRPV4 channels in addition to the previously reported expression of TRPV2 with a higher threshold of activating temperature.

Keyword

Mast cell; TRPV cation channels; TRPV4 protein; Temperature; Non-selective cation channel; Human

MeSH Terms

Allergens
Camphor
Capsaicin
Humans
Mast Cells
Membranes
Patch-Clamp Techniques
Phorbols
TRPV Cation Channels
Urticaria
Allergens
Camphor
Capsaicin
Phorbols
TRPV Cation Channels

Figure

• 

  Fig. 1. Heat-evoked currents observed at various ranges of temperature in HMC-1 cells. Current-voltage relations (I-V curves) obtained using the ramp-like pulses from –100 to 80 mV at room temperature (23∼25°C) and at different levels of increased temperature. The average values of normalized currents (pA/pF) are plotted against voltage. The responses to (MHT, 37∼39°C) are divided into two groups; relatively large increase weakly outward rectifying I/V curves (n=21, A) and small increase with linear I/V curves (n=19, B). (C) Summary of the responses to high temperature stimuli (HT, 44∼46°C, n=9) showing weakly outward rectifying I/V curves. (D) Remarkable increase of membrane conductance by very high temperature (VHT, 53∼55°C, n=3). (E) Summaries of the average densities of heat-activated current for the control, MHT, HT and VHT at –60. The data acquired from (B) were excluded in this statistics. ∗p<0.05; control versus MHT, HT and VHT, respectively.

• 

  Fig. 2. Desensitization of MHT-induced current and activation by 4aPDD in HMC-1 cells. (A) An exemplary current trace showing of repetitive application of MHT. Vertical lines reflect current responses to the repetitive ramp-like pulses from –100 to 80 mV. (B) Summary of I/V curves obtained by the ramp pulses, showing desensitization by repeated MHT stimuli (1st, 2nd, and 3rd, n=6) from room temperature (RT). The whole-cell current responded to repeated application of heat stimuli was measured using the perforated-patch recording mode. (C) Representative current trace showing the response to 1μM 4αPDD. (D) Summary of I-V curves obtained using the ramp-like pulse from –100 to 80 mV at room temperature, showing a weakly outward rectifying current induced by 1μM 4αPDD (n=12).

• 

  Fig. 3. Expression of TRPV2 and TRPV4 in HMC-1 cells. (A) RT-PCR analysis for TRPV1–4 in HMC-1 cells. Positive signals corresponding to the expected sizes of TRPV2 (573 b.p.) and TRPV4 (244 b.p.) were detected while not for TRPV1 (268 b.p.) and TRPV3 (772 b.p.). GAPDH was used as the control (rightmost lane). (B) and (C) Summary of the I-V curves obtained during the applications of 2μM capsaicin (n=4) or 2 mM camphor (n=3), respectively.

• 

  Fig. 4. Increase of intracellular Ca2+ concentration by heat and 4αPDD in HMC-1 cells. (A) Representative traces of F340/F380 showing the effect of 2μM 4αPDD at room temperature. The gray trace indicated by arrow is a representative negative response to 4αPDD. (B) Representative traces of normalized F340/F380 (see Methods), showing the sequential increases of [Ca2+]c by MHT and 2μM 4αPDD. The gray trace indicated by arrow is a representative negative response to 4αPDD with slight increase by MHT. (C) Summary of [Ca2+]c increases caused by 2μM 4αPDD and MHT in presence (left bar) and absence (middle bar) of 1.3 mM CaCl2. Number of tested cells are indicated in each bar.

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