State-Dependent Accessibility of the P-S6 Linker of Pacemaker (HCN) Channels Supports a Dynamic Pore-to-Gate Coupling Model

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 Kv 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 Cd2+ blockade (IC50 = 3–12 μM 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 (∆V1/2 = –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

Single-channel properties of ionic channel gated by cyclic nucleotides

This paper presents an extensive analysis of single-channel properties of cyclic nucleotide gated (CNG) channels, obtained by injecting into Xenopus laevis oocytes the mRNA encoding for the alpha and beta subunits from bovine rods. When the alpha and beta subunits of the CNG channel are coexpressed, at least three types of channels with different properties are observed. One type of channel has well-resolved, multiple conductive levels at negative voltages, but not at positive voltages. The other two types of channel are characterized by flickering openings, but are distinguished because they have a low and a high conductance. The alpha subunit of CNG channels has a well-defined conductance of about 28 pS, but multiple conductive levels are observed in mutant channels E363D and T364M. The conductance of these open states is modulated by protons and the membrane voltage, and has an activation energy around 44 kJ/mol. The relative probability of occupying any of these open states is independent of the cGMP concentration, but depends on extracellular protons. The open probability in the presence of saturating cGMP was 0.78, 0.47, 0.5, and 0.007 in the w.t. and mutants E363D, T364M, and E363G, and its dependence on temperature indicates that the thermodynamics of the transition between the closed and open state is also affected by mutations in the pore region. These results suggest that CNG channels have different conductive levels, leading to the existence of multiple open states in homomeric channels and to the flickering behavior in heteromeric channels, and that the pore is an essential part of the gating of CNG channels

Implications of metal exposure and liver function in Parkinsonian patients resident in the vicinities of ferroalloy plants

13accepted on 25-07-2009nonenoneSQUITTI R; GORGONE G; PANETTA V; LUCCHINI R; BUCOSSI S; ALBINI E; ALESSIO L; ALBERICI A; MELGARI JM; BENUSSI L; BINETTI G; ROSSINI PM; DRAICCHIO FSquitti, R; Gorgone, G; Panetta, V; Lucchini, Roberto; Bucossi, S; Albini, Elisa; Alessio, Lorenzo; Alberici, A; Melgari, Jm; Benussi, L; Binetti, G; Rossini, Pm; Draicchio, F

Cyclic nucleotide-gated channels. Molecular mechanisms of activation

Activation of cyclic nucleotide-gated (CNG) channels represents the final step in the transduction pathways in both vision and olfaction. Over the past several years, CNG channels have been found in a variety of other cell types where they might fulfill various physiological functions. The olfactory and photoreceptor CNG channels rely on the binding of at least two molecules of cAMP or cGMP at intracellular sites on the channel protein to open a nonspecific cation conductance with a significant permeability to Ca ions. A series of elegant experiments with cloned channels and chimeric constructs has revealed significant information regarding the binding and gating reactions that lead to CNG channel activation. These recent studies have identified several regions as well as specific amino acid residues distributed on the retinal or the olfactory CNG channel subunits that play a key role in channel regulation. In this review, we will focus on these specific molecular sites of activation and modulation of CNG channels