Atypical calcium regulation of the PKD2-L1 polycystin ion channel
- Howard Hughes Medical Institute, Boston Children's Hospital, United States
- Harvard Medical School, United States
Figures
Figure 1 with 1 supplement
PKD2-L1 channel deactivation is voltage-dependent.
(A) Diagram depicting symmetrical divalent free (DVF) conditions. (B) Top, voltage protocol used to generate tail currents. Representative currents from untransfected HEK cells (gray traces) and …
Figure 1—figure supplement 1
Decay of hyperpolarization-induced tail currents from PKD2-L1 channels.
(A) Single channel current events triggered by five hyperpolarization steps (top) in control conditions and after treatment with dibucaine. Dashed lines are 12.1 pA increments that separate multiple …
Figure 2 with 1 supplement
High intracellular Ca2+ irreversibly inactivates PKD2-L1.
(A) Representative PKD2-L1 currents captured at 1, 4 and 8 min time points recorded with the indicated buffered [free- Ca2+]; 2 mM Ca2+ in the external solution. (B) Time course of outward peak …
Figure 2—figure supplement 1
The time course of PKD2-L1 inactivation correlates with internal Ca2+ accumulation.
(A) Top, Voltage ramp applied at 0.5 Hz to activate PKD2-L1 currents expressed in HEK 293T cells. Bottom, Representative PKD2-L1 currents captured at 1, 4, and 8 min time points in physiological [Ca2…
Figure 3
Uncaging internal Ca2+ blocks outward single channel openings.
(A) Outward PKD2-L1 single channel events measured in the on-cell configuration; holding potential = 80 mV. Ca2+ dependent fluorescence was measured using Fluo-3; internal Ca2+ was caged with …
Figure 4 with 1 supplement
PKD2-L1 channel C-terminal truncations do not alter Ca2+-dependent inactivation.
(A) Cartoon depicting the locations of the C-terminal truncation mutants relative to the putative intracellular motifs. (B) Representative PKD2-L1 currents and the time courses of the potentiation …
Figure 4—figure supplement 1
Cell surface expression of the non-functional PKD2-L1 truncation mutants.
Transfected HEK 293T cells expressing N-terminally HA-tagged PKD2-L1 (HA-2L-1) and PKD2-L1 C-terminal truncation (HA-2-L1 Stop-588) were labeled with biotin. Total lysates and …
Figure 5 with 1 supplement
Block of PKD2L-1 by outward Ca2+ triggers channel inactivation.
(A) Left, Exemplar currents measured with the indicated [Ca2+]ex (inset). Right, resulting outward current densities at the indicated ECa (N = 6–7, Error ± SEM). (B) PKD2-L1 currents recorded from …
Figure 5—figure supplement 1
Outward PKD2-L1 current is blocked at membrane potentials positive to ECa.
(A) Left, Scheme depicting the ionic conditions and voltage protocol used to measure the voltage dependence of several TRP channels. Right, Whole-cell currents measured from HEK-293T cells …
Figure 6 with 1 supplement
Filter mutant D523N is not blocked by outward Ca2+ and is not inactivated.
(A) Amino acid alignment of the pore regions of PKD2 family members. The black bar indicates the selectivity filter. (B) Representative currents and time course of the average tail and peak current …
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Figure 6—source data 1
A table listing the relative permeabilities of cations through the PKD2-L1 channels as estimated by the measured reversal potentials (Erev).
Relative permeabilities (Px/PCs) of Na+, K+ and Ca2+ compared to Cs+ were calculated (see Methods) based on the measured reversal potential (N= 4–9, Error ± SEM).
- https://doi.org/10.7554/eLife.13413.013
Figure 6—figure supplement 1
The current-voltage relationships of PKD2-L1 channels in the presence of different extracellular cations.
Left, Representative current traces activated by 300 ms, +5 mV depolarizations from -100 to +100 mV; holding potential = −60 mV; recorded from the indicated PKD2-L1 channel/mutant. The external …
Figure 7 with 1 supplement
Loss of transition metal block in selectivity filter mutant, D523N.
(A, B) Effects of Zn2+ and Cd2+ on PKD2-L1 currents. Top, Exemplar PKD2-L1 currents activated by voltage ramps at the indicated [Zn2+]. Bottom, Corresponding time courses of the Zn2+ block of the Wt …
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Figure 7—source data 1
A table listing the potencies (IC50) of PKD2-L1 current antagonism by dibucaine and transition metals.
Concentration of half current inhibition (IC50) was estimated by fitting the concentration-percent current block relationship using the Hill equation (see Materials and methods).
- https://doi.org/10.7554/eLife.13413.016
Figure 7—figure supplement 1
Calmidazolium activation and dibucaine block is preserved in the D523N filter mutant channel.
(A) Left, Exemplar Wt and D523N currents activated by voltage ramps in the presence of two concentrations of the local anesthetic, dibucaine (Dbc). Right, Corresponding time course of current block. …
Figure 8
Proposed kinetic scheme of PKD2-L1 channel states and a hypothetical model of Ca2+ coordination sites in the selectivity filter.
(A) Calcium clamp of restricted spaces, such as primary cilia, by PKD2-L1. Resting [Ca2+] ≅ 500–700 nM in primary cilia. PKD2-L1 channels in the cilia membrane are potentiated before inactivation by …
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