Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed with a high temporal resolution (50-kHz filter) in symmetrical solutions with 50, 150, or 400 mM KCl and 2.5 mM CaCl2 and 2.5 mM MgCl2. At membrane potentials >+100 mV, the single-channel current showed a negative slope resistance, concomitantly with a flickery block, which was not influenced by Ca2+ or Mg2+. The analysis of the amplitude histograms by beta distributions revealed that current in this voltage range was reduced by two effects: rate limitation at the cytosolic side of the pore and gating with rate constants 10–20-fold higher than the cutoff frequency of the filter (i.e., dwell times in the microsecond range). The data were analyzed in terms of a model that postulates a coupling between both effects; if the voltage over the selectivity filter withdraws ions from the cavity at a higher rate than that of refilling from the cytosol, the selectivity filter becomes instable because of ion depletion, and current is interrupted by the resulting flickering. The fit of the IV curves revealed a characteristic voltage of 35 mV. In contrast, the voltage dependence of the gating factor R, i.e., the ratio between true and apparent single-channel current, could be fitted by exponentials with a characteristic voltage of 60 mV, suggesting that only part of the transmembrane potential is felt by the flux through the selectivity filter.

Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K channels, Kcv and Kcv. These small viral encoded K channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in Kcv and 40% in Kcv. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in Kcv. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels.

Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K+ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K+ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K+, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current.

Autonomic regulation of heart rate is largely mediated by the effect of cAMP on the pacemaker current I f , driven by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. cAMP 2 enhances HCN open probability by binding to the CNBD (cyclic nucleotide binding domain). The Clinker transmits the cAMP-induced conformational change from the CNBD to the pore and is thus considered a passive element in the opening transition. Here we report the finding of an allosteric binding site in the C-linker of HCN4 that implies a regulatory function of this domain. By structural and functional analysis we show that cyclic dinucleotides, an emerging class of second messengers in mammals, bind to this C-linker pocket (CLP) and antagonize cAMP regulation of HCN4 channels. Accordingly, cyclic dinucleotides prevent cAMP regulation of I f in sinoatrial-node myocytes, reducing heart rate by 30%. The same effect is attained by Compound 11, a molecule selected by virtual docking to the CLP. Occupancy of the CLP hence constitutes an efficient mechanism to prevent -adrenergic stimulation on I f . Our results highlight the regulative role of the C-linker in HCN4 and identify an isoform-specific drug target within the HCN family. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals, beyond their first reported role in innate immune system. IntroductionThe "funny" current (I f ) of cardiac pacemaker myocytes is an inward current activated by hyperpolarization of membrane voltage and controlled by intracellular cAMP 1 . Being activated and inhibited by -adrenergic and muscarinic M2 receptor stimulation, respectively, I f represents a basic physiological mechanism mediating autonomic regulation of heart rate and constitutes an ideal target for the pharmacological control of cardiac activity. The molecular determinants of I f are the Hyperpolarization-activated cAMP-gated (HCN) channels 2,3 . In these proteins, the transmembrane pore is connected at the N terminus to a voltage sensor domain and at the C-terminus to a cytosolic cyclic-nucleotide-binding domain (CNBD). The C-linker, anhelix folded domain of 90 amino acids, connects the CNBD to the pore. Structural studies showed that the cytosolic C-terminal fragment (C-linker + CNBD) assembles as a 4-fold symmetric tetramer in which the primary subunit interactions are provided by the linkers. The C-linkers form a ring in which the first two helices of one subunit (A' and B') form a helix-turn-helix motif that rests as an "elbow" on the "shoulder"formed by the second two helices, C' and D', of the neighboring subunit 4 . Enhancement of channel open probability by cAMP reflects the transition from the cAMP-unbound to the bound conformation of the 3 CNBD that induces a centrifugal rearrangement of the C-linkers with the shoulders twisting away from the elbows 5 . This movement in turn stabilizes the open conformation of the pore. Given the critical role of the Clinker in HCN channel modulation by ligands, it is interesting to note that this linker ...

The modular architecture of voltage-gated K+ (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates KvSynth1, a functional voltage-gated, outwardly rectifying K+ channel. KvSynth1 displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V1/2 = +56 mV; z of ∼1), including the depolarization-induced mode shift. The degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. This highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains.