Lab Anim Res.  2010 Dec;26(4):331-337. 10.5625/lar.2010.26.4.331.

Functional Analysis of TRPV6 Polymorphisms

• Kim BJ
• So I

• KMID: 2114700
• DOI: http://doi.org/10.5625/lar.2010.26.4.331

Abstract

The rate-limiting step of dietary calcium absorption in the intestine requires the brush border calcium entry channel transient receptor potential vanilloid 6 (TRPV6). The putatively-selected TRPV6 haplotype contains three candidate sites for functional differences, namely derived non-synonymous substitutions C157R, M378V and M681T. Functional electrophysiological characteristics between wild-type and mutant (C157R, M378V and M681T) TRPV6 proteins were investigated by cloning the mutant TRPV6 forms, transfecting cell lines, and carrying out electrophysiology experiments via patch clamp analysis. No statistically significant differences in biophysical channel function were found although one property, namely Ca2+-dependent inactivation, may show functionally-relevant differences between the wild-type and mutant TRPV6 proteins. This study shows that Ca2+-dependent inactivation is one of the good differentiation characteristics in TRPV6, and will be useful in an advancing our knowledge about TRPV6.

Keyword

Transient Receptor Potential; single nucleotide polymorphism; TRPV6

MeSH Terms

Absorption
Calcium
Calcium, Dietary
Cell Line
Clone Cells
Cloning, Organism
Electrophysiology
Haplotypes
Intestines
Lifting
Microvilli
Polymorphism, Single Nucleotide
Proteins
Calcium
Calcium, Dietary
Proteins

Figure

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  Figure 1. TRPV6WT is localized in endoplasmic reticulum (ER) in HEK 293 cells. HEK 293 cells were transiently transfected with GFP (A) or TRPV6-GFP (B) and fixed 48 hours after transfection. The maximum intensity projections (MIP) taken with the laser scanning microscope (LSM), reveal both plasma membrane and ER localization of TRPV6WT in HEK 293 cells. Bar=5 µm.

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  Figure 2. Current–voltage relationships (I-V curves) for the wild and mutant of TRPV6. A) IV curves for wild form obtained from the voltage ramps from −100 mV to +100 mV at the holding potential of −60 mV. Note the strong inward rectification in DVF and the shift of the reversal potentials when Ca2+ is the charge carrier (DVF: divalent free solution, Ca2+: 30 mM CaCl2 with NMDG+ as substitute for Na+). B) IV curves for C157R form, using the same protocol as in A. C) IV curves for M378V form, using the same protocol as in A. D) IV curves for M681T form, using the same protocol as in A.

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  Figure 3. I-V curves for the mutant of TRPV6. A) IV curves for C157R and M378V form obtained from the voltage ramps from −100 mV to +100 mV at the holding potential of −60 mV. Note the strong inward rectification in DVF and the shift of the reversal potentials when Ca2+ is the charge carrier (DVF: divalent free solution, Ca2+: 30 mM CaCl2 with NMDG+ as substitute for Na+). B) IV curves for C157R and M681T form, using the same protocol as in A. C) IV curves for M378V and M681T form, using the same protocol as in A. D) IV curves for C157R, M378V and M681T form, using the same protocol as in A.

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  Figure 4. Analyses of key feature of TRPV6: current density. A and B) Current density was measured at −100 mV under DVF and Ca2+ ionic conditions for each TRPV6. DVF: divalent free; Ca2+ refer to these divalent cations as the only charge carrier for the inward currents. Note that high current densities have been measured indicating high functional expression of the channels. C and D) Current densities were normalized to the current in DVF solution at −100 mV. There are no any differences among each TRPV6 forms. Data from between 4 and 5 cells for all graphs (W: wild; 1: C157R; 2: M378V; 3: M681T; 4: C157R and M378V; 5: C157R and M681T; 6: M378V and M681T; 7: C157R, M378V and M681T).

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  Figure 5. Analyses of key feature of TRPV6: Ca2+ dependent inactivation and block by Mg2+. A and B) Ca2+ dependent inactivation was estimated by the ratio of the currents at the end of the 400 ms voltage step and the maximal inward current during the step (I400ms, -100mV /Imax). C) Block of TRPV6 by Mg2+ was estimated from step protocols. I1 mM Mg2+, -100mV /IDVF, -100mV is the ratio of the maximal inward current measured from the same cell after changing from DVF to addition of 1 mM Mg2+. (1-ratio) gives the inhibition. Data from between 4 and 5 cells for all graphs (W: wild; 1: C157R; 2: M378V; 3: M681T; 4: C157R and M378V; 5: C157R and M681T; 6: M378V and M681T; 7: C157R, M378V and M681T). ∗P<0.05.

Reference

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