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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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Stoichiometric analyses of TRPML3/TRPV5 currents.(A–D) Traces show currents at −150 mV recorded from HEK293 cells expressing TRPV5/TRPML3, in the presence of extracellular solutions containing 140 mM Na+ (condition 1, black), 150 mM K+ (condition 2, red), 140 mM Na+, 0.1 mM EGTA (condition 3, blue), and 150 mM K+, 0.1 mM EGTA (condition 4, pink). We hypothesized that the current recorded under condition 1 represents the novel conductance, the current under condition 2 consists of the novel conductance and TRPML3, the current under condition 3 is composed of the novel conductance and TRPV5, and finally, under condition 4, we expected that the recorded current consists of all three conductances. (E–F) Subtraction of A from B (in E) which represents pure TRPML3; subtraction of A from C (in F) which results in pure TRPV5. (G–I) HEK293 cells were transfected with TRPML3 and TRPV5 expression plasmids either pure (0% and 100%) and at molar ratios 1∶10 (10%), 1∶5 (20%), 2∶3 (40%), 1∶1 (50%), 3∶2 (60%), 5∶1 (80%) and 10∶1 (90%), respectively. Currents were elicited at −150 mV under conditions 1–3 and individual conductances for TRPML3 and TRPV5 were extracted as described in (E–F). Shown is the average fraction of novel current (G, black circles), TRPML3 (H, red triangles), and TRPV5 (I, blue triangles) is plotted against the fraction of TRPML3 over TRPV5 (G,H) and TRPV5 over TRPML3 (I) (n = 5–11).
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pone-0058174-g004: Stoichiometric analyses of TRPML3/TRPV5 currents.(A–D) Traces show currents at −150 mV recorded from HEK293 cells expressing TRPV5/TRPML3, in the presence of extracellular solutions containing 140 mM Na+ (condition 1, black), 150 mM K+ (condition 2, red), 140 mM Na+, 0.1 mM EGTA (condition 3, blue), and 150 mM K+, 0.1 mM EGTA (condition 4, pink). We hypothesized that the current recorded under condition 1 represents the novel conductance, the current under condition 2 consists of the novel conductance and TRPML3, the current under condition 3 is composed of the novel conductance and TRPV5, and finally, under condition 4, we expected that the recorded current consists of all three conductances. (E–F) Subtraction of A from B (in E) which represents pure TRPML3; subtraction of A from C (in F) which results in pure TRPV5. (G–I) HEK293 cells were transfected with TRPML3 and TRPV5 expression plasmids either pure (0% and 100%) and at molar ratios 1∶10 (10%), 1∶5 (20%), 2∶3 (40%), 1∶1 (50%), 3∶2 (60%), 5∶1 (80%) and 10∶1 (90%), respectively. Currents were elicited at −150 mV under conditions 1–3 and individual conductances for TRPML3 and TRPV5 were extracted as described in (E–F). Shown is the average fraction of novel current (G, black circles), TRPML3 (H, red triangles), and TRPV5 (I, blue triangles) is plotted against the fraction of TRPML3 over TRPV5 (G,H) and TRPV5 over TRPML3 (I) (n = 5–11).

Mentions: To gain more insight into the interaction between TRPV5 and TRPML3, we transfected HEK293 cells with different ratios of plasmid DNA encoding the individual TRP proteins. We measured whole cell currents of the cotransfected cell populations under the four different conditions described above (Fig. 4A–D), and determined the contribution of individual conductances by subtracting the currents measured in the different conditions. For example, the contribution of TRPML3-based currents under condition 2 (Fig. 4B, at 150 mM K+) can be determined by subtracting the measured currents under condition 1 (Fig. 4A, at 140 mM Na+), resulting in TRPML3 (Fig. 4E). Subtracting condition 1 (Fig. 4A) from condition 3 (Fig. 4C), results in TRPV5 (Fig. 4F). Plotting the individual components for each current at different TRPV5:TRPML3 ratios revealed that the majority of the novel conductance was detected when the two channels were expressed at approximately equal levels (Fig. 4G). Shifting the ratio towards TRPML3 resulted in major contribution of TRPML3-based current of the total current (Fig. 4H), and expectedly, at a ratio that favors TRPV5, the main current of the total was TRPV5-based (Fig. 4I). Overall, if one of the two TRP channels was present at 80% or higher, very little of the new conductance was measured. These results suggest that the novel conductance requires a roughly equal contribution of TRPML3 and TRPV5.


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

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

Stoichiometric analyses of TRPML3/TRPV5 currents.(A–D) Traces show currents at −150 mV recorded from HEK293 cells expressing TRPV5/TRPML3, in the presence of extracellular solutions containing 140 mM Na+ (condition 1, black), 150 mM K+ (condition 2, red), 140 mM Na+, 0.1 mM EGTA (condition 3, blue), and 150 mM K+, 0.1 mM EGTA (condition 4, pink). We hypothesized that the current recorded under condition 1 represents the novel conductance, the current under condition 2 consists of the novel conductance and TRPML3, the current under condition 3 is composed of the novel conductance and TRPV5, and finally, under condition 4, we expected that the recorded current consists of all three conductances. (E–F) Subtraction of A from B (in E) which represents pure TRPML3; subtraction of A from C (in F) which results in pure TRPV5. (G–I) HEK293 cells were transfected with TRPML3 and TRPV5 expression plasmids either pure (0% and 100%) and at molar ratios 1∶10 (10%), 1∶5 (20%), 2∶3 (40%), 1∶1 (50%), 3∶2 (60%), 5∶1 (80%) and 10∶1 (90%), respectively. Currents were elicited at −150 mV under conditions 1–3 and individual conductances for TRPML3 and TRPV5 were extracted as described in (E–F). Shown is the average fraction of novel current (G, black circles), TRPML3 (H, red triangles), and TRPV5 (I, blue triangles) is plotted against the fraction of TRPML3 over TRPV5 (G,H) and TRPV5 over TRPML3 (I) (n = 5–11).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585263&req=5

pone-0058174-g004: Stoichiometric analyses of TRPML3/TRPV5 currents.(A–D) Traces show currents at −150 mV recorded from HEK293 cells expressing TRPV5/TRPML3, in the presence of extracellular solutions containing 140 mM Na+ (condition 1, black), 150 mM K+ (condition 2, red), 140 mM Na+, 0.1 mM EGTA (condition 3, blue), and 150 mM K+, 0.1 mM EGTA (condition 4, pink). We hypothesized that the current recorded under condition 1 represents the novel conductance, the current under condition 2 consists of the novel conductance and TRPML3, the current under condition 3 is composed of the novel conductance and TRPV5, and finally, under condition 4, we expected that the recorded current consists of all three conductances. (E–F) Subtraction of A from B (in E) which represents pure TRPML3; subtraction of A from C (in F) which results in pure TRPV5. (G–I) HEK293 cells were transfected with TRPML3 and TRPV5 expression plasmids either pure (0% and 100%) and at molar ratios 1∶10 (10%), 1∶5 (20%), 2∶3 (40%), 1∶1 (50%), 3∶2 (60%), 5∶1 (80%) and 10∶1 (90%), respectively. Currents were elicited at −150 mV under conditions 1–3 and individual conductances for TRPML3 and TRPV5 were extracted as described in (E–F). Shown is the average fraction of novel current (G, black circles), TRPML3 (H, red triangles), and TRPV5 (I, blue triangles) is plotted against the fraction of TRPML3 over TRPV5 (G,H) and TRPV5 over TRPML3 (I) (n = 5–11).
Mentions: To gain more insight into the interaction between TRPV5 and TRPML3, we transfected HEK293 cells with different ratios of plasmid DNA encoding the individual TRP proteins. We measured whole cell currents of the cotransfected cell populations under the four different conditions described above (Fig. 4A–D), and determined the contribution of individual conductances by subtracting the currents measured in the different conditions. For example, the contribution of TRPML3-based currents under condition 2 (Fig. 4B, at 150 mM K+) can be determined by subtracting the measured currents under condition 1 (Fig. 4A, at 140 mM Na+), resulting in TRPML3 (Fig. 4E). Subtracting condition 1 (Fig. 4A) from condition 3 (Fig. 4C), results in TRPV5 (Fig. 4F). Plotting the individual components for each current at different TRPV5:TRPML3 ratios revealed that the majority of the novel conductance was detected when the two channels were expressed at approximately equal levels (Fig. 4G). Shifting the ratio towards TRPML3 resulted in major contribution of TRPML3-based current of the total current (Fig. 4H), and expectedly, at a ratio that favors TRPV5, the main current of the total was TRPV5-based (Fig. 4I). Overall, if one of the two TRP channels was present at 80% or higher, very little of the new conductance was measured. These results suggest that the novel conductance requires a roughly equal contribution of TRPML3 and TRPV5.

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