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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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Whole-cell currents of cells expressing TRPV5, TRPML3, and both proteins in different ionic conditions.(A) Traces show representative currents obtained from non-transfected HEK293 cells, and cells expressing TRPV5, TRPML3, and TRPV5/TRPML3, in the presence of extracellular solutions starting with 140 mM Na+ (condition 1, black), and successively switched to 150 mM K+ (condition 2, red), 140 mM Na+, 0.1 mM EGTA (condition 3, blue), followed by 150 mM K+, 0.1 mM EGTA (condition 4, pink), and a final set of measurements conducted in condition 1. Currents were recorded during voltage steps from −150 mV to +130 mV in 20 mV increments, holding at 0 mV. (B) Mean values (±SD) of average inward current densities of TRPV5 (n = 10), TRPML3 (n = 12), and TRPV5/TRPML3 (n = 10) plotted against voltage in the presence of 140 Na+ (black circles), 150 K+ (red triangles), 140 Na+, 0.1 EGTA (blue triangles), and 150 K+, 0.1 EGTA (magenta diamonds), respectively. (C) Average inward current densities at −90 mV of HEK293 cells expressing the different channels in the four different conditions as indicated. Bar diagrams represent mean ± SD, numbers in parentheses are the number of cells analyzed. ** p<0.001 and * p<0.01, Student’s t-test, unpaired.
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pone-0058174-g002: Whole-cell currents of cells expressing TRPV5, TRPML3, and both proteins in different ionic conditions.(A) Traces show representative currents obtained from non-transfected HEK293 cells, and cells expressing TRPV5, TRPML3, and TRPV5/TRPML3, in the presence of extracellular solutions starting with 140 mM Na+ (condition 1, black), and successively switched to 150 mM K+ (condition 2, red), 140 mM Na+, 0.1 mM EGTA (condition 3, blue), followed by 150 mM K+, 0.1 mM EGTA (condition 4, pink), and a final set of measurements conducted in condition 1. Currents were recorded during voltage steps from −150 mV to +130 mV in 20 mV increments, holding at 0 mV. (B) Mean values (±SD) of average inward current densities of TRPV5 (n = 10), TRPML3 (n = 12), and TRPV5/TRPML3 (n = 10) plotted against voltage in the presence of 140 Na+ (black circles), 150 K+ (red triangles), 140 Na+, 0.1 EGTA (blue triangles), and 150 K+, 0.1 EGTA (magenta diamonds), respectively. (C) Average inward current densities at −90 mV of HEK293 cells expressing the different channels in the four different conditions as indicated. Bar diagrams represent mean ± SD, numbers in parentheses are the number of cells analyzed. ** p<0.001 and * p<0.01, Student’s t-test, unpaired.

Mentions: We next examined whether the suggested association of TRPML3 and TRPV5/6 channels becomes functionally manifest in whole cell patch clamp experiments using transfected HEK293 cells. For these analyses, we decided to focus on TRPML3 and TRPV5 and to investigate channel properties under four different conditions, which were selected to distinguish between TRPML3 and TRPV5 activities: under condition 1, both channels were expected to be inactive, condition 2 enables TRPML3 currents, condition 3 leads to activation of TRPV5, and condition 4 was picked for activation of both channels. Condition 1 is standard bath solution containing 140 mM Na+ as the major cation, 5 mM K+, and 1.5 mM Ca2+. Under this condition, TRPV5 and TRPML3 are inactive, and as expected, we did not record any considerable currents when either channel was expressed alone (Fig. 2A,B). In cells cotransfected with TRPV5 and TRPML3 expression plasmids, however, we were able to elicit inwardly rectified currents when we stepped to negative voltages. At −150 mV, in standard bath solution, we measured a current density of −0.129±0.052 nA/pF (mean±SD, n = 10) for this novel current. Condition 2 contained K+ as the major cation (150 mM K+), a low concentration of Na+ (2 mM), and 1.5 mM Ca2+. As expected, TRPV5 was inactive in this condition and TRPML3 displayed inwardly rectifying currents as shown previously [3], [18] with a current density of −0.10±0.006 nA/pF (n = 12) at −150 mV. Cotransfected cells exhibited larger currents than TRPML3-only expressing cells with an average current density of −0.169±0.067 nA/pF (n = 10) at −150 mV. Under condition 3, containing 140 mM Na+, 5 mM K+, and 0.1 mM EGTA, which favors active TRPV5 and inactive TRPML3, we measured TRPV5 activity at −150 mV of −0.54±0.12 nA/pF (n = 10); TRPML3 was inactive, as expected. Cotransfected cells displayed average currents of −0.326±0.007 nA/pF (n = 10) at −150 mV. Finally, under condition 4, containing 150 mM K+, 2 mM Na+, and 0.1 mM EGTA, which enables TRPV5 and TRPML3 to be active, we measured average current densities at −150 mV for TRPV5 of −0.379±0.1 nA/pF, for TRPML3 of 0.07±0.02 nA/pF, and for the cotransfected cells: −0.283±0.09 nA/pF (n = 10, 12, and 10, respectively). Our whole cell recording experiments show that HEK293 cells cotransfected with expression plasmids for TRPV5 and TRPML3 display a novel conductance that is active under conditions where the homomeric channels are inactive.


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

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

Whole-cell currents of cells expressing TRPV5, TRPML3, and both proteins in different ionic conditions.(A) Traces show representative currents obtained from non-transfected HEK293 cells, and cells expressing TRPV5, TRPML3, and TRPV5/TRPML3, in the presence of extracellular solutions starting with 140 mM Na+ (condition 1, black), and successively switched to 150 mM K+ (condition 2, red), 140 mM Na+, 0.1 mM EGTA (condition 3, blue), followed by 150 mM K+, 0.1 mM EGTA (condition 4, pink), and a final set of measurements conducted in condition 1. Currents were recorded during voltage steps from −150 mV to +130 mV in 20 mV increments, holding at 0 mV. (B) Mean values (±SD) of average inward current densities of TRPV5 (n = 10), TRPML3 (n = 12), and TRPV5/TRPML3 (n = 10) plotted against voltage in the presence of 140 Na+ (black circles), 150 K+ (red triangles), 140 Na+, 0.1 EGTA (blue triangles), and 150 K+, 0.1 EGTA (magenta diamonds), respectively. (C) Average inward current densities at −90 mV of HEK293 cells expressing the different channels in the four different conditions as indicated. Bar diagrams represent mean ± SD, numbers in parentheses are the number of cells analyzed. ** p<0.001 and * p<0.01, Student’s t-test, unpaired.
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Related In: Results  -  Collection

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pone-0058174-g002: Whole-cell currents of cells expressing TRPV5, TRPML3, and both proteins in different ionic conditions.(A) Traces show representative currents obtained from non-transfected HEK293 cells, and cells expressing TRPV5, TRPML3, and TRPV5/TRPML3, in the presence of extracellular solutions starting with 140 mM Na+ (condition 1, black), and successively switched to 150 mM K+ (condition 2, red), 140 mM Na+, 0.1 mM EGTA (condition 3, blue), followed by 150 mM K+, 0.1 mM EGTA (condition 4, pink), and a final set of measurements conducted in condition 1. Currents were recorded during voltage steps from −150 mV to +130 mV in 20 mV increments, holding at 0 mV. (B) Mean values (±SD) of average inward current densities of TRPV5 (n = 10), TRPML3 (n = 12), and TRPV5/TRPML3 (n = 10) plotted against voltage in the presence of 140 Na+ (black circles), 150 K+ (red triangles), 140 Na+, 0.1 EGTA (blue triangles), and 150 K+, 0.1 EGTA (magenta diamonds), respectively. (C) Average inward current densities at −90 mV of HEK293 cells expressing the different channels in the four different conditions as indicated. Bar diagrams represent mean ± SD, numbers in parentheses are the number of cells analyzed. ** p<0.001 and * p<0.01, Student’s t-test, unpaired.
Mentions: We next examined whether the suggested association of TRPML3 and TRPV5/6 channels becomes functionally manifest in whole cell patch clamp experiments using transfected HEK293 cells. For these analyses, we decided to focus on TRPML3 and TRPV5 and to investigate channel properties under four different conditions, which were selected to distinguish between TRPML3 and TRPV5 activities: under condition 1, both channels were expected to be inactive, condition 2 enables TRPML3 currents, condition 3 leads to activation of TRPV5, and condition 4 was picked for activation of both channels. Condition 1 is standard bath solution containing 140 mM Na+ as the major cation, 5 mM K+, and 1.5 mM Ca2+. Under this condition, TRPV5 and TRPML3 are inactive, and as expected, we did not record any considerable currents when either channel was expressed alone (Fig. 2A,B). In cells cotransfected with TRPV5 and TRPML3 expression plasmids, however, we were able to elicit inwardly rectified currents when we stepped to negative voltages. At −150 mV, in standard bath solution, we measured a current density of −0.129±0.052 nA/pF (mean±SD, n = 10) for this novel current. Condition 2 contained K+ as the major cation (150 mM K+), a low concentration of Na+ (2 mM), and 1.5 mM Ca2+. As expected, TRPV5 was inactive in this condition and TRPML3 displayed inwardly rectifying currents as shown previously [3], [18] with a current density of −0.10±0.006 nA/pF (n = 12) at −150 mV. Cotransfected cells exhibited larger currents than TRPML3-only expressing cells with an average current density of −0.169±0.067 nA/pF (n = 10) at −150 mV. Under condition 3, containing 140 mM Na+, 5 mM K+, and 0.1 mM EGTA, which favors active TRPV5 and inactive TRPML3, we measured TRPV5 activity at −150 mV of −0.54±0.12 nA/pF (n = 10); TRPML3 was inactive, as expected. Cotransfected cells displayed average currents of −0.326±0.007 nA/pF (n = 10) at −150 mV. Finally, under condition 4, containing 150 mM K+, 2 mM Na+, and 0.1 mM EGTA, which enables TRPV5 and TRPML3 to be active, we measured average current densities at −150 mV for TRPV5 of −0.379±0.1 nA/pF, for TRPML3 of 0.07±0.02 nA/pF, and for the cotransfected cells: −0.283±0.09 nA/pF (n = 10, 12, and 10, respectively). Our whole cell recording experiments show that HEK293 cells cotransfected with expression plasmids for TRPV5 and TRPML3 display a novel conductance that is active under conditions where the homomeric channels are inactive.

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