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State-dependent accessibility of the P-S6 linker of pacemaker (HCN) channels supports a dynamic pore-to-gate coupling model.
Siu CW
,
Azene EM
,
Au KW
,
Lau CP
,
Tse HF
,
Li RA
.
Abstract
The hyperpolarization-activated cyclic nucleotide-modulated channel gene family (HCN1-4) encodes the membrane depolarizing current that underlies pacemaking. Although the topology of HCN resembles K(v) channels, much less is known about their structure-function correlation. Previously, we identified several pore residues in the S5-P linker and P-loop that are externally accessible and/or influence HCN gating, and proposed an evolutionarily conserved pore-to-gate mechanism. Here we sought dynamic evidence by assessing the functional consequences of Cys-scanning substitutions in the unexplored P-S6 linker (residues 352-359), the HCN1-R background (that is, resistant to sulfhydryl-reactive agents). None of A352C, Q353C, A354C, P355C, V356C, S357C, M358C, or S359C produced functional currents; the loss-of-function of Q353C, A354C, S357C, and M358C could be rescued by the reducing agent dithiothreitol. Q353C, A354C, and S357C, but not M358C and HCN1-R, were sensitive to Cd(2+) blockade (IC(50) = 3-12 microM vs. >1 mM). External application of the positively charged covalent sulfhydryl modifier MTSET irreversibly reduced I (-140mV) of Q353C and A354C to 27.9 +/- 3.4% and 58.2 +/- 13.1% of the control, respectively, and caused significant steady-state activation shifts (DeltaV(1/2) = -21.1 +/- 1.6 for Q353C and -10.0 +/- 2.9 mV for A354C). Interestingly, MTSET reactivity was also state dependent. MTSET, however, affected neither S357C nor M358C, indicating site specificity. Collectively, we have identified novel P-S6 residues whose extracellular accessibility was sterically and state dependent and have provided the first functional evidence consistent with a dynamic HCN pore-to-gate model.
Fig. 1. Schematic representation of HCN1-R. The six transmembrane segments of a monomeric subunit of the HCN1-R are shown. Six endogenous cysteine residues are removed together with the truncation of the âCOOH terminus. Five of the six remaining cysteine residues are replaced by a serine, threonine, or isoleucine substitution. C303 in the S5 segment is left in the final HCN1-R construct because of lack of functional current upon mutations. Their approximate locations, as well as the GYG signature motif, are highlighted as shown. The eight amino acid residues in the P-S6 motif of the final HCN1-R construct are individually replaced by cysteine and named accordingly
Fig. 2. a Representative current tracings of WT HCN1 and HCN1-R. Currents were elicited by stepping to a family of 3-s electrical pulses ranging from 0 to â140 mV in 10-mV increments from a holding potential of â30 mV. b Steady-state currentâvoltage (IâV) relationships (at 3 s). c Steady-state activation curves
Fig. 3. Representative current tracings of the eight P-S6 Cys-substituted constructs (a) before and (b) after DTT. c Steady-state activation curves of Q353C, A354C, S357C, and M358C after DTT treatment
Fig. 4. Effects of Cd2+ on HCN1-R, Q353C, A354C, S357C, and M358C channels. a Representative current tracings measured at â140 mV in the absence and presence of Cd2+ as indicated. b Doseâresponse relationships
Fig. 5. Representative current tracings of Q353C, A354C, S357C, and M358C before and after external application of MTSET
Fig. 6. a Steady-state IâV relationships of S357C and M358C before and after MTSET. b Steady-state activation curves. c Tail IâV relationships
Fig. 7. a Steady-state IâV relationships of Q353C and A354C before and after MTSET. b Steady-state activation curves. c Tail IâV relationships
Fig. 8. Time course of the development of current inhibition at â140 mV upon external application of 2.5 mM MTSET with a stimulation frequency of 0.03 and 0.016 Hz to a Q353C and b A354C channels. c, d The same experimental data from A and B are plotted as normalized Iâ140mV against the pulse number (rather than time)
Fig. 9. Western blot analysis of the membrane proteins isolated from oocytes injected with cRNA of WT HCN1, HCN1-R, and eight pore Cys-substituted constructs under control and reducing conditions
Fig. 10. Schematic representation of the HCN pore summarizing our present results and those of our two previously published studies. Gray circles, cysteinyl side chain; open circles, permeant ion; filled circles, endogenous amino acid
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