Strengthen our conclusion on theJ.G.J.Hoenderop et al.Fig. six. Cd2 sensitivity of TRPV5, TRPV6 and TRPV5D542A mono and multimers. (A ) Current oltage relationships obtained during voltage ramps in nominally divalentfree extracellular options in the absence and presence of 2, 20, 200 and 2000 mM CdCl2 for cells transfected with (A) TRPV5555, (B) TRPV5D542A, (C) TRPV55D542A55, (D) a mixture of TRPV5 and TRPV5D542A in a 3:1 ratio, (E) a mixture of TRPV6666 and TRPV5D542A within a 1:1 ratio and (F) a mixture of TRPV666 and TRPV5D542A within a 1:1 ratio. (G ) Dose esponse curves for the effect of Cd2 measured at 00 mV. (G) Dose esponse curves for TRPV5555 and TRPV5D542A. From Hill functions ted towards the information (solid curves), we obtained values for KD and nHill of 64 nM and 0.78, respectively, for TRPV5555 compared with 313 mM and 0.84 for TRPV5D542A. Note that the Cd2 sensitivity on the TRPV5555 concatemer was not signi antly various from that with the TRPV5 monomers (KD = 74 nM, nHill = 0.81; data not shown). (H) Dose esponse curves for TRPV55D542A55 and also the mixture of TRPV5 and TRPV5D542A. From a Hill function ted to the TRPV55D542A55 information (strong curve), we obtained values for KD and nHill of 1.0 mM and 0.77, respectively. The data for the mixture of TRPV5 and TRPV5D542A were not effectively ted by a SC66 Epigenetics single Hill function, indicating a population of channels with distinct Cd2 sensitivities. (I) Dose esponse curves for TRPV6666 and for mixtures of TRPV5D542A with TRPV6666 and TRPV666, respectively. From a Hill function ted to the TRPV6666 data, values for KD and nHill of 163 nM and 1.05, respectively, had been obtained. Equivalent values have been obtained for TRPV666 (KD = 157 nM, nHill = 0.93) and for the TRPV6 monomer (KD = 261 nM, nHill = 1.05). The dose esponse curve for the mixture of TRPV6666 and TRPV5D542A was well described by the weighted sum of the Hill functions for TRPV6666 and TRPV5D542A (strong curve). In contrast, the dose esponse curve for the mixture of TRPV666 and TRPV5D542A was poorly ted by the weighted sum on the Hill functions for TRPV6666 and TRPV5D542A (dotted line).tetrameric stoichiometry with the channel. Wildtype TRPV5 and TRPV6 display voltagedependent opening with the channel upon hyperpolarization, and deactivation upon depolarization, that is illustrated for TRPV5555 in Figure 7B. The apparent open probability with the channel as a function of voltage, which was determined as the normalized inward present upon stepping to 00 mV from unique test potentials, didn’t differ signi antly amongst TRPV5555, TRPV666, TRPV6666 and monomeric TRPV5 or TRPV6 constructs (Figure 7G; data not shown). This voltage dependence, which depends upon intracellular Mg2 (Voets et al., 2001), is abolished in the TRPV5D542A mutant (Figure 7C and G). Interestingly, the effect of this mutation on voltagedependent gating appears to be dominant, considering the fact that mutating only a single subunit in a tetrameric TRPV5 construct (TRPV55D542A55) resulted in voltageindependent currents (Figure 7D and H). Likewise, coexpression on the TRPV666 construct with TRPV5D542A led to voltageindependent currents, consistent with formation of a TRPV666TRPV5D542A tetrameric channel (Figure 7E and H). In contrast, voltagedependent gating was lowered, but not abolished, in cells coexpressing TRPV6666 or TRPV5555 with TRPV5D542A, indicating formation of separate voltagedependent TRPV6666 (or TRPV5555) channels and voltageindependent tetrameric TRPV5D542A channels (Figure 7F and H). Taken together, th.
