The large chlorella virus PBCV-1, which contains double-stranded DNA (dsDNA), encodes a 94-codon open reading frame (ORF) that contains a motif resembling the signature sequence of the pore domain of potassium channel proteins. Phylogenetic analyses of the encoded protein, Kcv, indicate a previously unidentified type of potassium channel. The messenger RNA encoded by the ORF leads to functional expression of a potassium-selective conductance in Xenopus laevis oocytes. The channel blockers amantadine and barium, but not cesium, inhibit this conductance, in addition to virus plaque formation. Thus, PBCV-1 encodes the first known viral protein that functions as a potassium-selective channel and is essential in the virus life cycle.

'Funny-' (f-) channels of cardiac sino-atrial node (SAN) cells are key players in the process of pacemaker generation and mediate the modulatory action of autonomic transmitters on heart rate. The molecular components of f-channels are the hyperpolarization-activated, cyclic nucleotidegated (HCN) channels. Of the four HCN isoforms known, two (HCN4 and HCN1) are expressed in the rabbit SAN at significant levels. However, the properties of f-channels of SAN cells do not conform to specific features of the two isoforms expressed locally. For example, activation kinetics and cAMP sensitivity of native pacemaker channels are intermediate between those reported for HCN1 and HCN4. Here we have explored the possibility that both HCN4 and HCN1 isoforms contribute to the native I f in SAN cells by co-assembling into heteromeric channels. To this end, we used heterologous expression in human embryonic kidney (HEK) 293 cells to investigate the kinetics and cAMP response of the current generated by co-transfected (HCN4 + HCN1) and concatenated (HCN4-HCN1 (4-1) tandem or HCN1-HCN4 (1-4) tandem) rabbit constructs and compared them with those of the native f-current from rabbit SAN. 4-1 tandem, but not cotransfected, currents had activation kinetics approaching those of I f ; however, the activation range of 4-1 tandem channels was more negative than that of the f-channel and their cAMP sensitivity were poorer (although that of 1-4 tandem channels was normal). Co-transfection of 4-1 tandem channels with minK-related protein 1(MiRP1) did not alter their properties. HCN1 and HCN4 may contribute to native f-channels, but a 'context'-dependent mechanism is also likely to modulate the channel properties in native tissues.

Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation “delay” by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between “reluctant” and “willing” states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.

Background: HCN2 and HCN4 respond to cAMP, whereas HCN1 does not. Results: The C-linker plus CNBD of HCN2 and HCN4 show cAMP-induced tetramerization, whereas that of HCN1 contains prebound cAMP and is tetrameric. Conclusion: HCN1 does not respond to the addition of cAMP because its CNBD contains cAMP already. Significance: Tetramerization of the C terminus controls ligand gating in HCN channels.

cAMP signaling in the brain mediates several higher order neural processes. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels directly bind cAMP through their cytoplasmic cyclic nucleotide binding domain (CNBD), thus playing a unique role in brain function. Neuronal HCN channels are also regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit that antagonizes the effects of cAMP by interacting with the channel CNBD. To unravel the molecular mechanisms underlying the dual regulation of HCN channel activity by cAMP/ TRIP8b, we determined the NMR solution structure of the HCN2 channel CNBD in the cAMP-free form and mapped on it the TRIP8b interaction site. We reconstruct here the full conformational changes induced by cAMP binding to the HCN channel CNBD. Our results show that TRIP8b does not compete with cAMP for the same binding region; rather, it exerts its inhibitory action through an allosteric mechanism, preventing the cAMP-induced conformational changes in the HCN channel CNBD.

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels in the brain associate with their auxiliary subunit TRIP8b (also known as PEX5R), a cytoplasmic protein expressed as a family of alternatively spliced isoforms. Recent in vitro and in vivo studies have shown that association of TRIP8b with HCN subunits both inhibits channel opening and alters channel membrane trafficking, with some splice variants increasing and others decreasing channel surface expression. Here, we address the structural bases of the regulatory interactions between mouse TRIP8b and HCN1. We find that HCN1 and TRIP8b interact at two distinct sites: an upstream site where the C-linker/cyclic nucleotide-binding domain of HCN1 interacts with an 80 aa domain in the conserved central core of TRIP8b; and a downstream site where the C-terminal SNL (Ser-Asn-Leu) tripeptide of the channel interacts with the tetratricopeptide repeat domain of TRIP8b. These two interaction sites play distinct functional roles in the effects of TRIP8b on HCN1 trafficking and gating. Binding at the upstream site is both necessary and sufficient for TRIP8b to inhibit channel opening. It is also sufficient to mediate the trafficking effects of those TRIP8b isoforms that downregulate channel surface expression, in combination with the trafficking motifs present in the N-terminal region of TRIP8b. In contrast, binding at the downstream interaction site serves to stabilize the C-terminal domain of TRIP8b, allowing for optimal interaction between HCN1 and TRIP8b as well as for proper assembly of the molecular complexes that mediate the effects of TRIP8b on HCN1 channel trafficking.

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The present palette of opsin-based optogenetic tools lacks a light-gated potassium (K(+)) channel desirable for silencing of excitable cells. Here, we describe the construction of a blue-light-induced K(+) channel 1 (BLINK1) engineered by fusing the plant LOV2-Jα photosensory module to the small viral K(+) channel Kcv. BLINK1 exhibits biophysical features of Kcv, including K(+) selectivity and high single-channel conductance but reversibly photoactivates in blue light. Opening of BLINK1 channels hyperpolarizes the cell to the K(+) equilibrium potential. Ectopic expression of BLINK1 reversibly inhibits the escape response in light-exposed zebrafish larvae. BLINK1 therefore provides a single-component optogenetic tool that can establish prolonged, physiological hyperpolarization of cells at low light intensities.