The cytosolic region of LRMP regulates HCN4 but does not antagonize cAMP binding.

A: Voltage dependence of activation for HCN4 alone (black) or co-transfected with LRMP 1-479Cit (red) in the presence or absence of 1 mM intracellular cAMP (open symbols). B: Average (± standard error of the mean) midpoints of activation for HCN4 in the absence or presence of LRMP 1-479Cit and/or 1 mM cAMP using the same colour scheme as A. C: Average (± standard error of the mean) time constants of deactivation for HCN4 in the absence or presence of LRMP 1-479Cit and/or 1mM cAMP using the same colour scheme as A. Small circles in B and C represent individual cells and values in parentheses are the number of independent recordings for each condition. * indicates a significant (P < 0.05) difference. All means, standard errors, and exact P-values are in Table 1.

Midpoints of Activation for HCN Channel Constructs

The pre-coiled-coil region of the LRMP N-terminus is necessary and sufficient to regulate HCN4.

A-D: Voltage-dependence of activation for HCN4 in the absence (black) or presence (red) of LRMP 1-227 (A), LRMP 228-539 (B), LRMP 1-108 (C), or LRMP 110-230 (D), and/or 1mM intracellular cAMP (open symbols). The midpoints of activation for HCN4 with (dotted line) or without (solid line) 1 mM cAMP in the absence of LRMP are shown. A inset: Schematic of LRMP showing the predicted coiled-coil domain (CCD) and ER-transmembrane and luminal domains (ER). E: Average (± standard error of the mean) midpoints of activation for HCN4 in the absence or presence of LRMP constructs and/or 1mM cAMP using the same colour scheme as A-D. Small circles represent individual recordings and values in parentheses are the number of independent recordings for each condition. * indicates a significant (P < 0.05) difference. All means, standard errors, and exact P-values are in Table 2.

Midpoints of Activation in HCN4 in the Presence of LRMP Fragments

The distal HCN4 N-terminus is required for functional regulation by LRMP.

A-D: Voltage-dependence of activation for HCN4 Δ1-25 (A), HCN4 Δ1-62 (B), HCN4 Δ1-130 (C), and HCN4 Δ1-185 (D) in the absence (black) or presence of LRMP (red) and/or 1mM intracellular cAMP (open symbols). E: Average (± standard error of the mean) midpoints of activation for HCN4 Δ1-25, HCN4 Δ1-62, HCN4 Δ1-130, and HCN4 Δ1-185 in the absence or presence of LRMP and/or 1mM cAMP using the same colour scheme as A. Small circles represent individual recordings and values in parentheses are the number of independent recordings for each condition. * indicates a significant (P < 0.05) difference. All means, standard errors, and exact P-values are in Table 1.

The HCN4 C-terminus is not the primary site for functional regulation by LRMP.

A: Voltage-dependence of activation for HCN4 S719X in the absence (black) or presence of LRMP (red) and/or 1mM intracellular cAMP (open symbols). B: Average (± standard error of the mean) midpoints of activation for HCN4 S719X in the absence or presence of LRMP and/or 1mM cAMP using the same colour scheme as B. C: Voltage-dependence of activation for HCN4 V604X in the absence or presence of LRMP or 1mM intracellular cAMP using the same colour scheme as B. D: Average (± standard error of the mean) midpoints of activation for HCN4 V604X in the absence or presence of LRMP or 1mM cAMP using the same colour scheme as B. Small circles represent individual recordings in B and D and values in parentheses are the number of independent recordings for each condition. * indicates a significant (P < 0.05) difference. All means, standard errors, and exact P-values are in Tables 1 and 3.

Acceptor Photobleaching FRET Between LRMP and HCN Channel Fragments

The N-terminus of LRMP FRETs with the N-terminus of HCN4

A: Average (± standard error of the mean) acceptor photobleaching FRET efficiency between Citrine or the Citrine-tagged N-terminal region of the LRMP and Cerulean-tagged fragments of HCN4. The dotted line is the average FRET in YFP-CFP concatemers from a prior study (Wang et al., 2020a). B: Average (± standard error of the mean) acceptor photobleaching FRET efficiency between Citrine-tagged fragments of the LRMP N-terminus and Cerulean-tagged fragments of HCN4 or HCN2. Small circles in A and B represent individual recordings and values in parentheses are the number of independent recordings for each condition. * indicates a significant (P < 0.05) difference compared to control FRET in cells co-transfected with Citrine and with Cerulean-tagged HCN4 N-terminal fragments. All means, standard errors, and exact P-values are in Table 3. C: Schematic representations of the Citrine-tagged LRMP fragments and Cerulean-tagged HCN4 and HCN2 fragments used in FRET experiments.

Mutants in the HCN4 C-linker disrupt LRMP’s functional effects.

A: Structure of the HCN4 C-linker (PDB: 7NP4) and S4-S5 linker overlapped with HCN1 (PDB: 5U6P; beige; top left) or showing key residues (top right). Alignments of the HCN2 and HCN4 C-linker sequences with the HCN4 P545/T547 sites highlighted in red (bottom). B: Voltage-dependence of activation for HCN4 P545A/T547F (PT/AF) in the absence (black) or presence of LRMP (red) and/or 1mM intracellular cAMP (open symbols). C: Average (± standard error of the mean) midpoints of activation for HCN4 PT/AF in the absence or presence of LRMP and/or 1mM cAMP using the same colour scheme as B. D: Voltage-dependence of activation for HCN2 A467P/F469T (AF/PT) in the absence or presence of LRMP and/or 1mM intracellular cAMP using the same colour scheme as B. E: Average (± standard error of the mean) midpoints of activation for HCN2 AF/PT in the absence or presence of LRMP and/or 1mM cAMP using the same colour scheme as B. Small circles represent individual recordings in C and E and values in parentheses are the number of independent recordings for each condition. * indicates a significant (P < 0.05) difference. All means, standard errors, and exact P-values are in Table 1.

The HCN4 N-terminus confers LRMP regulation on HCN2

A: Voltage-dependence of activation for HCN4-2 (HCN4 1-518 + HCN2 442-863) in the absence (black) or presence of LRMP (red) and/or 1mM intracellular cAMP (open symbols). B: Average (± standard error of the mean) midpoints of activation for HCN4-2 in the absence or presence of LRMP and/or 1mM cAMP using the same colour scheme as A. C: Voltage-dependence of activation for HCN4-2 VVGPT (HCN4 1-212 + HCN2 135-863 M338V/C341V/S345G/A467P/F469T) in the absence or presence of LRMP and/or 1mM intracellular cAMP using the same colour scheme as A. D: Average (± standard error of the mean) midpoints of activation for HCN4-2 VVGPT in the absence or presence of LRMP and/or 1mM cAMP using the same colour scheme as A. Insets: Schematics of the HCN4-2 (A) and HCN4-2 VVGPT (C) channels with HCN4 sequence shown in black and HCN2 in blue. Small circles represent individual recordings in B and D and values in parentheses are the number of independent recordings for each condition. * indicates a significant (P < 0.05) difference. All means, standard errors, and exact P-values are in Table 1.

Model of interactions between HCN4 and LRMP

A: Structure of the cAMP-transduction centre in HCN4 formed by the C-linker (yellow), S4-S5 linker (teal), and HCNDs of two subunits (purple; PDB: 7NP4). B: Schematic of the proposed interactions between LRMP (red) and the HCN4 channel. The cytoplasmic N-terminus of LRMP interacts with the HCN4 N-terminus to functionally regulate cAMP-dependent shifts in activation at the interface between the N-terminus, C-linker, and S4-S5 linker. Made with BioRender.

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DNA Constructs Used in this Study