Abstract
Numerical model-based simulations provide important insights into ion channel gating when experimental limitations exist. Here, a novel strategy combining numerical simulations with patch clamp experiments was used to investigate the net positive charges in the putative transmembrane segment 4 (S4) of the atypical, positively-shifted voltage-dependence of polycystic kidney disease 2-like 1 (PKD2L1) channel. Charge-neutralising mutations (K452Q, K455Q and K461Q) in S4 reduced gating charges, positively shifted the Boltzmann-type activation curve [i.e., open probability (Popen)-V curve] and altered the time-courses of activation/deactivation of PKD2L1, indicating that this region constitutes part of a voltage sensor. Numerical reconstruction of wild-type (WT) and mutant PKD2L1-mediated currents necessitated, besides their voltage-dependent gating parameters, a scaling factor that describes the voltage-dependence of maximal conductance, Gmax. Subsequent single-channel conductance (γ) measurements revealed that voltage-dependence of Gmax in WT can be explained by the inward-rectifying property of γ, which is greatly changed in PKD2L1 mutants. Homology modelling based on PKD2 and NaVAb structures suggest that such voltage dependence of Popen and γ in PKD2L1 could both reflect the charged state of the S4 domain. The present conjunctive experimental and theoretical approaches provide a framework to explore the undetermined mechanism(s) regulating TRP channels that possess non-classical voltage-dependent properties.
Highlights
Numerical model-based simulations provide important insights into ion channel gating when experimental limitations exist
Homology modelling based on PKD2 and NaVAb structures suggest that such voltage dependence of Popen and γ in polycystic kidney disease 2-like 1 (PKD2L1) could both reflect the charged state of the segment 4 (S4) domain
Recent structural analyses of KV1.2 and PKD2 suggest that the S4–S5 linker interacts with the C-terminus of S6, and mechanical movement of this linker leads to constriction or dilatation of the channel pore[24, 25]
Summary
To investigate the functional impact of charge-neutralisation, we tested the voltage dependency of mutant PKD2L1 channels using the same step-pulse protocol as above (Fig. 4a). K461Q showed Boltzmann-type voltage-dependent activation similar to WT, but with a pronounced positive shift in V0.5 (Fig. 4b and c) and decreased voltage-sensitivity (i.e., reduced valence Zapp; Fig. 4d) These results strongly suggest that the charged state of the S4 region is crucial for PKD2L1 gating. The model was modified to fit the voltage-dependence of mutant PKD2L1 channels that could not be fully activated even by extreme depolarisation (Fig. 5). One plausible mechanism for this apparent voltage-dependence of maximal conductance is the intrinsic rectifying property or partial opening (or dilation) of an ion conductive pore[32,33,34] To examine this possibility more directly, we measured the unitary conductance (γ) of constitutively active WT and mutant PKD2L1 channels by means of single channel recording. The above results imply that the three positively charged K452, K455 and K461 residues in the putative S4 region of the PKD2L1 channel may differentially contribute to its voltage-dependent activation through at least two distinct mechanisms, i.e., alteration of Popen and rectification of unitary ionic flow or the voltage-dependence of γ
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