Critical intra-linker interactions of HCN1-encoded pacemaker channels revealed by interchange of S3–S4 determinants

Mechanistic Role of I f Revealed by Induction of Ventricular Automaticity by Somatic Gene Transfer of Gating-Engineered Pacemaker (HCN) Channels

“…Ad-CGI-HCN1-Ins-induced I f had positively (V 1/2 ϭϪ60.4Ϯ0.7 mV; kϭ8.7Ϯ0.6; nϭ7) and negatively (V 1/2 ϭϪ87.7Ϯ0.7 mV; kϭ11.4Ϯ0.4; nϭ17) shifted activation midpoints, respectively ( Figure 2E), consistent with our previous heterologous expression experiments. 22,25 The differences in activation threshold Note that the kinetics observed here in LVCMs were faster than the same channels that we reported previously. 23 Nonetheless, the difference in activation midpoints relative to WT persisted (see E).…”

“…EVY235-7⌬⌬⌬ and Ins encode HCN1 channels in which the S3-S4 linkers have been shortened by deleting residues 235 to 237 and lengthened by inserting 2 glutamines to flank each of the C-and N-terminal sides of residues 235 to 237 (ie, QQ235 to 237QQ) to favor and inhibit opening, respectively, as recently described. 22,23,25 Figure 1 shows that I K1 ( Figure 1A and 1E), which could be completely blocked by 1 mmol/L Ba 2ϩ ( Figure 1B and 1F), was robustly expressed in control (nontransduced or Ad-CGItransduced) adult guinea pig LVCMs (nϭ20 from 4 animals). For Ad-CGI-HCN1-transduced cells, a similar Ba 2ϩ -sensitive I K1 with properties not different from those of control cells also was expressed (Figure 1C and 1E; PϾ0.05).…”

“…22,23 Although HCN4 is the predominant isoform expressed in the SA node (at least in rabbit), 24 HCN1 was chosen because its structurefunction properties were investigated more extensively in our previous studies 13,22,23,[25][26][27][28][29][30] and the library of constructs available. However, because HCN channels were engineered to exhibit particular gating profiles for our experiments, the ultimate biophysical properties of a given recombinant channel override the specific species and isoform used.…”

jShaw1, a low-threshold, fast-activating Kv3 from the hydrozoan jellyfish Polyorchis penicillatus

“…The length of this loop has a significant effect on voltage sensitivity in the K v 1 channels (Gonzalez et al, 2000;Gonzalez et al, 2001;Mathur et al, 1997), HCN channels (Henrikson et al, 2003;Lesso and Li, 2003;Tsang et al, 2004) and L-type Ca ++ channels (Nakai et al, 1994). It is possible that the shorter loop constrains the relative movement of the C-terminal end of the S3 helix and the N-terminal end of the S4 helix in such a way that energetically favors an open conformation, contributing to the fact that jShaw1 has a more hyperpolarized activation curve that the two mouse K v 3 channels.…”

Bioartificial Sinus Node Constructed via In Vivo Gene Transfer of an Engineered Pacemaker HCN Channel Reduces the Dependence on Electronic Pacemaker in a Sick-Sinus Syndrome Model

“…20 Thus, native I f is difficult to reproduce by simple expression of a single HCN isoform. Here, we took advantage of the engineered construct HCN1-EVY235-7⌬⌬⌬ (or HCN1-⌬⌬⌬) channels, the S3-S4 linker of which has been systematically shortened by deleting residues 235 to 237 to favor channel opening 21,22 and thereby compensate for any context-dependent gating effects. We conjectured that overexpressing EVY235-7⌬⌬⌬ channels alone in atrial or ventricular CMs can sufficiently mimic the heteromultimeric native nodal I f without the need for simultaneous manipulation of the expression levels of multiple HCN isoforms and/or other modifying subunits and factors that may be present in nodal but not muscle cells.…”