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A novel ion channel formed by interaction of TRPML3 with TRPV5.

Guo Z, Grimm C, Becker L, Ricci AJ, Heller S - PLoS ONE (2013)

Bottom Line: It has pharmacological similarity with TRPML3 and requires functional TRPML3 as well as functional TRPV5.Single channel analyses revealed that TRPML3 and TRPV5 heteromers have different features than the respective homomers, and furthermore, that they occur in potentially distinct stoichiometric configurations.Based on overlapping expression of TRPML3 and TRPV5 in the kidney and the inner ear, we propose that TRPML3 and TRPV5 heteromers could have a biological function in these organs.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology-HNS, Stanford University School of Medicine, Palo Alto, California, United States of America.

ABSTRACT
TRPML3 and TRPV5 are members of the mucolipin (TRPML) and TRPV subfamilies of transient receptor potential (TRP) cation channels. Based on sequence similarities of the pore forming regions and on structure-function evidence, we hypothesized that the pore forming domains of TRPML and TRPV5/TRPV6 channels have similarities that indicate possible functional interactions between these TRP channel subfamilies. Here we show that TRPML3 and TRPV5 associate to form a novel heteromeric ion channel. This novel conductance is detectable under conditions that do not activate either TRPML3 or TRPV5. It has pharmacological similarity with TRPML3 and requires functional TRPML3 as well as functional TRPV5. Single channel analyses revealed that TRPML3 and TRPV5 heteromers have different features than the respective homomers, and furthermore, that they occur in potentially distinct stoichiometric configurations. Based on overlapping expression of TRPML3 and TRPV5 in the kidney and the inner ear, we propose that TRPML3 and TRPV5 heteromers could have a biological function in these organs.

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Effects of dominant-negative and inactive isoforms of TRPML3 and TRPV5.(A) Representative whole cell currents shown were obtained from cells coexpressing TRPML3D458K and TRPV5 (ML3D458K/ V5), TRPV5D535K and TRPV5 (V5D535K/ V5), TRPML3 and TRPV5D535K (ML3/V5D535K), TRPV5D550K and TRPV5 (V5D550K/ V5), and TRPML3 and TRPV5D550K (ML3/V5D550K) under the four conditions indicated. Currents were recorded during voltage steps from −150 mV to +130 mV in 20 mV increments, holding at 0 mV. (B) Average inward current densities at −150 mV of the various TRPML3 and TRPV5 channel pairs under the four different conditions as indicated. Bar diagrams represent mean±SD, numbers in parentheses are the number of cells analyzed. (C) Pharmacological properties of HEK293 cells expressing TRPV5 and TRPML3/TRPV5D550K (ML3/V5D550K). Representative traces were recorded during voltage steps from 0 mV to −150 mV under condition 3 before (blue) and after (cyan) application of 100 µM ruthenium red (RR). (D) Quantitative analysis of the percentage of inhibition at −150 mV (mean±SD, n = parenthesized).
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pone-0058174-g005: Effects of dominant-negative and inactive isoforms of TRPML3 and TRPV5.(A) Representative whole cell currents shown were obtained from cells coexpressing TRPML3D458K and TRPV5 (ML3D458K/ V5), TRPV5D535K and TRPV5 (V5D535K/ V5), TRPML3 and TRPV5D535K (ML3/V5D535K), TRPV5D550K and TRPV5 (V5D550K/ V5), and TRPML3 and TRPV5D550K (ML3/V5D550K) under the four conditions indicated. Currents were recorded during voltage steps from −150 mV to +130 mV in 20 mV increments, holding at 0 mV. (B) Average inward current densities at −150 mV of the various TRPML3 and TRPV5 channel pairs under the four different conditions as indicated. Bar diagrams represent mean±SD, numbers in parentheses are the number of cells analyzed. (C) Pharmacological properties of HEK293 cells expressing TRPV5 and TRPML3/TRPV5D550K (ML3/V5D550K). Representative traces were recorded during voltage steps from 0 mV to −150 mV under condition 3 before (blue) and after (cyan) application of 100 µM ruthenium red (RR). (D) Quantitative analysis of the percentage of inhibition at −150 mV (mean±SD, n = parenthesized).

Mentions: Our results thus far are compatible with two scenarios. Either, TRPV5 and TRPML3 form a heteromeric channel in which both TRP proteins contribute to the channel pore or, the novel conductance is TRPML3-based and simply modulated (activated) by close association with TRPV5. To gain more insight into these two possible scenarios, we investigated several mutant isoforms of TRPML3 and TRPV5 and coexpressed them with the wild type channels in equal ratios. TRPML3(D458K) is a dominant-negative isoform of TRPML3 [3], [4], and when co-expressed with TRPML3, it abolishes all TRPML3-based currents (Fig. 5B: ML3/ML3D458K). When coexpressed with TRPV5, we neither recorded channel activity in TRPML3-enabling condition 2 (as expected), nor did we detect the novel current under condition 1 (Fig. 5A,B: ML3D458K/V5). TRPV5-based currents were still measurable under conditions 3 and 4. These results suggest that functional TRPML3 is needed for the novel conductance. It also shows that dominant-negative TRPML3, when expressed in equal ratio with TRPV5 is not able to abolish presumptive TRPV5 homomeric channel activity. Overall, this result is compatible with both scenarios.


A novel ion channel formed by interaction of TRPML3 with TRPV5.

Guo Z, Grimm C, Becker L, Ricci AJ, Heller S - PLoS ONE (2013)

Effects of dominant-negative and inactive isoforms of TRPML3 and TRPV5.(A) Representative whole cell currents shown were obtained from cells coexpressing TRPML3D458K and TRPV5 (ML3D458K/ V5), TRPV5D535K and TRPV5 (V5D535K/ V5), TRPML3 and TRPV5D535K (ML3/V5D535K), TRPV5D550K and TRPV5 (V5D550K/ V5), and TRPML3 and TRPV5D550K (ML3/V5D550K) under the four conditions indicated. Currents were recorded during voltage steps from −150 mV to +130 mV in 20 mV increments, holding at 0 mV. (B) Average inward current densities at −150 mV of the various TRPML3 and TRPV5 channel pairs under the four different conditions as indicated. Bar diagrams represent mean±SD, numbers in parentheses are the number of cells analyzed. (C) Pharmacological properties of HEK293 cells expressing TRPV5 and TRPML3/TRPV5D550K (ML3/V5D550K). Representative traces were recorded during voltage steps from 0 mV to −150 mV under condition 3 before (blue) and after (cyan) application of 100 µM ruthenium red (RR). (D) Quantitative analysis of the percentage of inhibition at −150 mV (mean±SD, n = parenthesized).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585263&req=5

pone-0058174-g005: Effects of dominant-negative and inactive isoforms of TRPML3 and TRPV5.(A) Representative whole cell currents shown were obtained from cells coexpressing TRPML3D458K and TRPV5 (ML3D458K/ V5), TRPV5D535K and TRPV5 (V5D535K/ V5), TRPML3 and TRPV5D535K (ML3/V5D535K), TRPV5D550K and TRPV5 (V5D550K/ V5), and TRPML3 and TRPV5D550K (ML3/V5D550K) under the four conditions indicated. Currents were recorded during voltage steps from −150 mV to +130 mV in 20 mV increments, holding at 0 mV. (B) Average inward current densities at −150 mV of the various TRPML3 and TRPV5 channel pairs under the four different conditions as indicated. Bar diagrams represent mean±SD, numbers in parentheses are the number of cells analyzed. (C) Pharmacological properties of HEK293 cells expressing TRPV5 and TRPML3/TRPV5D550K (ML3/V5D550K). Representative traces were recorded during voltage steps from 0 mV to −150 mV under condition 3 before (blue) and after (cyan) application of 100 µM ruthenium red (RR). (D) Quantitative analysis of the percentage of inhibition at −150 mV (mean±SD, n = parenthesized).
Mentions: Our results thus far are compatible with two scenarios. Either, TRPV5 and TRPML3 form a heteromeric channel in which both TRP proteins contribute to the channel pore or, the novel conductance is TRPML3-based and simply modulated (activated) by close association with TRPV5. To gain more insight into these two possible scenarios, we investigated several mutant isoforms of TRPML3 and TRPV5 and coexpressed them with the wild type channels in equal ratios. TRPML3(D458K) is a dominant-negative isoform of TRPML3 [3], [4], and when co-expressed with TRPML3, it abolishes all TRPML3-based currents (Fig. 5B: ML3/ML3D458K). When coexpressed with TRPV5, we neither recorded channel activity in TRPML3-enabling condition 2 (as expected), nor did we detect the novel current under condition 1 (Fig. 5A,B: ML3D458K/V5). TRPV5-based currents were still measurable under conditions 3 and 4. These results suggest that functional TRPML3 is needed for the novel conductance. It also shows that dominant-negative TRPML3, when expressed in equal ratio with TRPV5 is not able to abolish presumptive TRPV5 homomeric channel activity. Overall, this result is compatible with both scenarios.

Bottom Line: It has pharmacological similarity with TRPML3 and requires functional TRPML3 as well as functional TRPV5.Single channel analyses revealed that TRPML3 and TRPV5 heteromers have different features than the respective homomers, and furthermore, that they occur in potentially distinct stoichiometric configurations.Based on overlapping expression of TRPML3 and TRPV5 in the kidney and the inner ear, we propose that TRPML3 and TRPV5 heteromers could have a biological function in these organs.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology-HNS, Stanford University School of Medicine, Palo Alto, California, United States of America.

ABSTRACT
TRPML3 and TRPV5 are members of the mucolipin (TRPML) and TRPV subfamilies of transient receptor potential (TRP) cation channels. Based on sequence similarities of the pore forming regions and on structure-function evidence, we hypothesized that the pore forming domains of TRPML and TRPV5/TRPV6 channels have similarities that indicate possible functional interactions between these TRP channel subfamilies. Here we show that TRPML3 and TRPV5 associate to form a novel heteromeric ion channel. This novel conductance is detectable under conditions that do not activate either TRPML3 or TRPV5. It has pharmacological similarity with TRPML3 and requires functional TRPML3 as well as functional TRPV5. Single channel analyses revealed that TRPML3 and TRPV5 heteromers have different features than the respective homomers, and furthermore, that they occur in potentially distinct stoichiometric configurations. Based on overlapping expression of TRPML3 and TRPV5 in the kidney and the inner ear, we propose that TRPML3 and TRPV5 heteromers could have a biological function in these organs.

Show MeSH
Related in: MedlinePlus