Figure 4

Specific amino acid replacements in the HLH domain and S2-S3 linker correlate with the fast inactivation phenotype. (A) Amino acid sequence alignment of the HLH domain. Conserved residues in TRPV5/6 (pink), TRPV5-type (light green) and TRPV6-type (dark green) channels are highlighted. (B) Structure of one subunit of the human TRPV6 channel (hTRPV6; PDB: 6E2F)²² showing the structural triad HLH domain (blue), S2-S3 linker (yellow) and TD-helix (purple). TM region: Transmembrane region. The inset shows the location of the signature side chains in TRPV5. (C) Amino acid sequence alignment of the S2-S3 linker of the TRPV5-type, TRPV6-type and TRPV5/6 channels. Non-inactivating channels show conserved residues (light green) that differ from residues present in mammalian TRPV6 channels (dark green). Red stars indicate residues located at the S2-S3 linker previously mutated by Nilius et al.¹⁷, affecting fast inactivation in a mammalian TRPV6 channel. (D) Normalized whole cell current traces recorded from transiently transfected HEK-293T cells expressing hTRPV5, hTRPV6, and engineered TRPV5 (hTRPV5_SS; E288D; F292L; S298T) containing the three-residue signature sequence at the HLH domain of TRPV6 channels. (E) Normalized whole cell current traces recorded from the chicken TRPV5 (chTRPV5_WT) and its triple mutant (chTRPV5_SS; E290D; F294L; S300T) containing the three-residue signature sequence at the HLH domain of TRPV6 channels and human TRPV6 channel (hTRPV6 WT). (F) Normalized whole cell current traces recorded from the spotted gar TRPV5/6 (gTRPV5/6_WT) and its mutant (chTRPV5/6_SS; V294L; C300T) containing the three-residue signature sequence at the HLH domain of TRPV6 channels and human TRPV6 channel (hTRPV6 WT). (G) Pooled data comparing the residual currents (see methods). (H) Pooled data comparing the inactivation time constants for inactivating clones (see methods). All current traces were recorded in the presence of 2 mM extracellular Ca²⁺ and in response to a voltage pulse at −160 mV for 60 ms from Vh=0 mV (Panels D; E; F). Black line indicates the fitting to a single exponential function (D,E,F). All bars represent mean value, errors represent S.E. and white dots represent values for each experiment (Panels G; H). ** represents p = 0.01 and *p = 0.05.