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.