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SummaryThe KtrAB ion transporter is a complex of the KtrB membrane protein and KtrA, an RCK domain. RCK domains regulate eukaryotic and prokaryotic membrane proteins involved in K+ transport. Conflicting functional models have proposed two different oligomeric arrangements for RCK domains, tetramer versus octamer. Our results for the KtrAB RCK domain clearly show an octamer in solution and in the crystal. We determined the structure of this protein in three different octameric ring conformations that resemble the RCK-domain octamer observed in the MthK potassium channel but show striking differences in size and symmetry. We present experimental evidence for the association between one RCK octameric ring and two KtrB membrane proteins. These results provide insights into the quaternary organization of the KtrAB transporter and its mechanism of activation and show that the RCK-domain octameric ring model is generally applicable to other ion-transport systems.

EAG, ERG and ELK are members of the Ether-a-go-go voltage-gated potassium channel family (1). Each channel possesses a C-terminal domain that is homologous to the cyclic nucleotide-binding domain of CNG/HCN channels but insensitive to cyclic nucleotides (2,3). X-ray crystallography of EAG suggests that the binding pocket of this region, termed the cyclic nucleotide-binding homology domain (CNBhD), is intrinsically “liganded” by the side chains of two residues, Y699 and L701 (4,8). To test this hypothesis, Y699 and L701 were substituted with alanines (AA) to mimic an “unliganded” state. AA mutant currents expressed in Xenopus oocytes showed the 20-80% rise time of activation was decreased by more than three times, concordant with very slow HCN channel activation in the absence of cyclic nucleotides (5). Homology models based on HCN and EAG crystal structures (6,7) revealed a possible salt bridge, as found in HCN channels (8). Charge reversals in two of the corresponding residues in EAG (R535 and D606) independently slowed channel activation, and charge swapping restored normal activation, indicative of a salt bridge. Alanine mutations at both sites preserved the WT activation, as if the salt bridge is not required for normal communication between the liganded conformation of the CNBhD and the gating machinery. However, the very slow phenotype of the unliganded CNBhD conformation was disrupted by the salt bridge perturbation in the R535A-AA and D606A-AA mutants and phenocopied the full CNBhD deletion. Thus, the salt bridge appears to mediate interaction between the unliganded CNBhD and the gating machinery. Supported by NIH NS081320.1. PNAS 91:3438 (1994); 2. Neuropharmacology 35:841 (1996); 3. JBC 284:27989 (2009); 4. Nature 481:530 (2012); 5. Nature. 351:145 (1991); 6. Nature 425:200 (2003); 7. JMB 423:34 (2012); 8. JGP 124: 663 (2004)

Here we describe the initial functional characterization of a cyclic nucleotide regulated ion channel from the bacterium Mesorhizobium loti and present two structures of its cyclic nucleotide binding domain, with and without cAMP. The domains are organized as dimers with the interface formed by the linker regions that connect the nucleotide binding pocket to the pore domain. Together, structural and functional data suggest the domains form two dimers on the cytoplasmic face of the channel. We propose a model for gating in which ligand binding alters the structural relationship within a dimer, directly affecting the position of the adjacent transmembrane helices.

In drosophila, the tetrameric Ether-a-go-go (EAG) voltage-gated K+ channel contains in its C-terminal cytoplasmic region a substrate-like binding domain for Ca2+/calmodulin-dependent protein kinase II (CaMKII-BD). This Ser/Thr kinase forms a dodecamer and each subunit includes an association domain mediating multimerization and a catalytic domain corresponding to the typical protein kinase fold. In between these domains, CaMKII contains a regulatory segment (RS) harboring a CaMKII-BD with homology to that of EAG followed by a Ca2+/CaM-binding region. This RS has a regulatory role, mediating the inhibition and activation of the kinase. CaMKII is activated by Ca2+/CaM-binding and can become Ca2+/CaM-independent upon phosphorylation of its CaMKII-BD. EAG is phosphorylated by CaMKII in vivo, and the two proteins form a stable complex in vitro. CaMKII-bound EAG keeps the kinase active in the absence of Ca2+/CaM and autophosphorylation.The interaction of the constitutively active CaMKII kinase domain with the EAG CaMKII-BD has been characterized. The measurement of KD values showed that the EAG CaMKII-BD is a high affinity CaMKII substrate, lying in the nanomolar range in comparison with the micromolar range of typical protein kinase substrates. Crystal structures were determined for the ternary complexes CaMKII-Mg2+/ATP-EAG and CaMKII-Mg2+/ADP-EAGP corresponding to the initial and final states of the trans-phosphorylation mechanism, respectively. These structures are globally very similar, with relative rearrangements at the catalytic site. An alanine scan of the EAG residues lying at the CaMKII-EAG interface revealed that practically every residue contributes significantly for the binding strength of the complex. The association of the two proteins in the cell might lie beyond the usual transient kinase-substrate association model, likely corresponding to a long-lasting robust complex.

The effect of ascorbic acid, light, and storage on the stability of the pigments beta-carotene and capsanthin in red pepper (Capsicum annuum) powder has been elucidated by determining the amount of pigment in samples treated by various concentrations of ascorbic acid. Determination of pigment concentration has been performed after different storage times using high-performance liquid chromatography. The dependence of the concentration of pigments on the concentration of ascorbic acid, presence of light and the storage time has been assessed by stepwise regression analysis. The concentration of pigments decreased at longer storage time and increased at higher concentration of ascorbic acid, beta-carotene being more sensitive towards storage time and concentration of ascorbic acid than capsanthin. Interaction between the effects of light and storage time, and light and concentration of ascorbic acid has been established.

Many voltage-dependent K+ channels open when the membrane is depolarized and then rapidly close by a process called inactivation. Neurons use inactivating K+ channels to modulate their firing frequency. In Shaker-type K+ channels, the inactivation gate, which is responsible for the closing of the channel, is formed by the channel's cytoplasmic amino terminus. Here we show that the central cavity and inner pore of the K+ channel form the receptor site for both the inactivation gate and small-molecule inhibitors. We propose that inactivation occurs by a sequential reaction in which the gate binds initially to the cytoplasmic channel surface and then enters the pore as an extended peptide. This mechanism accounts for the functional properties of K+ channel inactivation and indicates that the cavity may be the site of action for certain drugs that alter cation channel function.

TTR Met30 Familial Amyloidotic Polyneuropathy of the Portuguese type (FAP) is an incapacitating and lethal hereditary disorder that affects predominantly young adults of both genders. Portuguese type FAP patients have sensory, motor and autonomic polyneuropathy. The generalised systemic amyloid infiltration involves the heart, leading to the characteristic granular bright sparkling echocardiographic pattern. LV wall thickening occurs in the late phases of the disease. LV diastolic dysfunction has been reported in the absence of systolic dysfunction; an abnormal diastolic transmitral flow pattern assessed by pulsed wave Doppler (PW) was described. PW is very much dependent on load conditions. Tissue Doppler imaging (TDI) has been used as a more reliable method to assess long axis diastolic function.1--To identify the incremental value of TDI in the assessment of diastolic function in FAP. 2--To correlate diastolic pattern abnormalities and left ventricular mass index (LVMI) in FAP patients.We performed a prospective evaluation of 24 consecutive FAP patients and selected 14 (sinus rhythm, age45 years). Diastolic function was assessed by PW and classified as normal (GI-E/A1) or abnormal (GII-E/A1). TDI was performed in 4 sites of the mitral annulus (septum, lateral, inferior, anterior). Velocities of the rapid filling wave (E') and atrial contraction wave (A') were measured and E'/A' calculated. In each site we considered the TDI as normal (E'/A'1) or abnormal (E'/A'1). The LVMI was calculated by Devereux's formula.Age, gender and heart rate were similar in both groups. TDI at the septal mitral annulus was normal in all of the GI patients (E'/A': 1.29 +/- 0.19) and suggestive of abnormal LV relaxation in all of the GII patients (E'/A': 0.82 +/- 0.11, p0.0001). TDI revealed abnormal diastolic pattern when a restricted number of sites of the mitral annulus were assessed, even in GI patients and before PW abnormalities occurred. Fractional shortening (FS) and LVMI were similar in GI and GII (FS-GI: 45.5 +/- 5.3, GII 43.5 +/- 8.1%, p: NS; LVMI--GI: 66 +/- 9.3, GII: 67 +/- 3.0 g/m2 p: NS).The assessment of mitral annulus motion has introduced new data in the study of diastolic function of FAP patients. An abnormal LV relaxation pattern occurred early in the evolution of the disease in patients with normal LVMI and systolic function.

The assessment of the mitral annulus motion with tissue Doppler imaging is claimed to be an accurate method to quantify global left ventricular systolic and diastolic function. However, it is not yet perfectly defined which site of the annulus must be selected. Familial amyloidotic polyneuropathy of the Portuguese type (FAP) is an hereditary systemic disease in which diastolic dysfunction may occur.1--To determine if in FAP patients the mitral annulus motion is independent of the selected site. 2--To compare pulsed wave Doppler parameters with tissue Doppler parameters in the different annular sites.Of 24 FAP patients studied, 14 were included. In each patient we performed conventional transmitral pulsed wave Doppler and tissue Doppler in the 4 sites of the mitral annulus and measured the velocities of the rapid filling wave e, of the atrial contraction wave a and calculated e/a ratio.According to the transmitral inflow profile, patients were divided in 2 groups: Group I--normal global diastolic function and Group II--abnormal relaxation. Group I--33% of these patients showed e/a1 in the four sites and 67% showed e/a1 in at least 1, but not in all the sites. The rate of normal sites per patient was 3.1. Group II--25% of these patients showed e/a1 in the 4 sites of the annulus and 75% had e/a1 in at least 1, but not in all the sites analysed. The rate of abnormal sites/patient was 3.1. in this group. When conventional and tissue Doppler data were compared (bland and altman) the septal portion of the annulus was the one with the best correlation.1--The assessment of the mitral annulus motion with tissue Doppler imaging is dependent on the site selected for study. 2--The septal site was the one that showed the highest correlation and concordance between pulsed wave Doppler and tissue Doppler. 3--The relative number of normal versus abnormal sites was determinant of the transmitral pattern. 4--Tissue Doppler imaging identified: a) among patients until now classified as normal diastolic function, a subgroup of patients with abnormal function in some sites of the annulus and b) among patients with abnormal relaxation, a subgroup with normal diastolic function in some sites of the annulus.

In the last decades, the conceptual and technical advances in paediatric cardiology have brought additional options concerning the provision of paediatric cardiology care. The purpose of this study was to analyse the initial experience of a paediatric cardiology service in a conventional general hospital serving a population of nearly 450,000 inhabitants, mostly Chinese. Access to several levels of care, including cooperation with a tertiary referral center, is described. From September 1993 to July 1995, 717 children with either suspected or diagnosed heart disease were seen: 294 (41%) were discharged and, of the remaining 423 with heart disease, 364 (85%) had congenital heart disease; 75% were adequately managed with the available local resources. Appropriate referrals provided more advanced approaches for the smaller group requiring them. The training of local doctors has ensured the continuity of a good standard of care for this group of paediatric patients in Macau, with an evident reduction in direct as well as indirect costs.

The HERG voltage-dependent K+ channel plays a role in cardiac electrical excitability, and when defective, it underlies one form of the long QT syndrome. We have determined the crystal structure of the HERG K+ channel N-terminal domain and studied its role as a modifier of gating using electrophysiological methods. The domain is similar in structure to a bacterial light sensor photoactive yellow protein and provides the first three-dimensional model of a eukaryotic PAS domain. Scanning mutagenesis of the domain surface has allowed the identification of a hydrophobic "hot spot" forming a putative interface with the body of the K+ channel to which it tightly binds. The presence of the domain attached to the channel slows the rate of deactivation. Given the roles of PAS domains in biology, we propose that the HERG N-terminal domain has a regulatory function.

The PDZ domain, also known as the GLGF repeat/DHR domain, is an approximately 90-amino acid motif discovered in a recently identified family of proteins termed MAGUKs (membrane-associated guanylate kinase homologues). Sequence comparison analysis has since identified PDZ domains in over 50 proteins. Like SH2 and SH3 domains, the PDZ domains mediate specific protein-protein interactions, whose specificities appear to be dictated by the primary structure of the PDZ domain as well as its binding target. Using recombinant fusion proteins and a blot overlay assay, we show that a single copy of the PDZ domain in human erythrocyte p55 binds to the carboxyl terminus of the cytoplasmic domain of human erythroid glycophorin C. Deletion mutagenesis of 21 amino acids at the amino terminus of the p55 PDZ domain completely abrogates its binding activity for glycophorin C. Using an alanine scan and surface plasmon resonance technique, we identify residues in the cytoplasmic domain of glycophorin C that are critical for its interaction with the PDZ domain. The recognition specificity of the p55 PDZ domain appears to be unique, since the three PDZ domains of hDlg (human lymphocyte homologue of the Drosophila discs large tumor suppressor) do not bind the cytoplasmic domain of glycophorin C. Taken together with our previous studies, these results complete the identification of interacting domains in the ternary complex between p55, glycophorin C, and protein 4.1. Implications of these findings are discussed in terms of binding specificity and the regulation of cytoskeleton-membrane interactions.

DNA gyrase is a type II DNA topoisomerase from bacteria that introduces supercoils into DNA1,2. It catalyses the breakage of a DNA duplex (the G segment), the passage of another segment (the T segment) through the break, and then the reunification of the break. This activity involves the opening and closing of a series of molecular ‘gates’ which is coupled to ATP hydrolysis. Here we present the crystal structure of the ‘breakage–reunion’ domain of the gyrase at 2.8 A resolution. Comparison of the structure of this 59K (relative molecular mass, 59,000) domain with that of a 92K fragment of yeast topoisomerase II (ref. 3) reveals a very different quaternary organization, and we propose that the two structures represent two principal conformations that participate in the enzymatic pathway. The gyrase structure reveals a new dimer contact with a grooved concave surface for binding the G segment and a cluster of conserved charged residues surrounding the active-site tyrosines. It also shows how breakage of the G segment can occur and, together with the topoisomerase II structure, suggests a pathway by which the T segment can be released through the second gate of the enzyme. Mutations that confer resistance to the quinolone antibacterial agents cluster at the new dimer interface, indicating how these drugs might interact with the gyrase–DNA complex.

KCNH channels form an important family of voltage gated potassium channels. These channels include a N-terminal Per-Arnt-Sim (PAS) domain with unknown function. In other proteins PAS domains are implicated in cellular responses to environmental queues through small molecule binding or involvement in signaling cascades. To better understand their role we characterized the structural properties of several channel PAS domains. We determined high resolution structures of PAS domains from the mouse EAG (mEAG), drosophila ELK (dELK) and human ERG (hERG) channels and also of the hERG domain without the first nine amino acids. We analyzed these structures for features connected to ligand binding and signaling in other PAS domains. In particular, we have found cavities in the hERG and mEAG structures that share similarities with the ligand binding sites from other PAS domains. These cavities are lined by polar and apolar chemical groups and display potential flexibility in their volume. We have also found that the hydrophobic patch on the domain β-sheet is a conserved feature and appears to drive the formation of protein-protein contacts. In addition, the structures of the dELK domain and of the truncated hERG domain revealed the presence of N-terminal helices. These helices are equivalent to the helix described in the hERG NMR structures and are known to be important for channel function. Overall, these channel domains retain many of the PAS domain characteristics known to be important for cell signaling.

The Ktr transport system belongs to the large superfamily of Trk/Ktr/HKT ion transporters that share structural similarities with potassium and sodium channels. The KtrAB complex is composed by a dimer of the membrane-integrant KtrB and an octameric ring of the cytosolic KtrA protein, which is an RCK-domain. Each KtrB subunit has a K+ selective pore. We tested several nucleotides for eluting KtrA from a high-affinity resin and found that ADP and ATP are the most potent suggesting higher affinities towards the protein. The crystal structure of the complex, recently solved by our group, revealed that the ring conformation of KtrA changes upon nucleotide binding. To determine the functional effects of these nucleotides we reconstituted the KtrAB complex from B. subtilis into liposomes and measured the transport activity reflected by uptake of 86Rb+. Our results show that ATP activates the transporter, while the flux mediated by KtrAB-ADP is similar to the flux of KtrB reconstituted without KtrA. Increasing amounts of extraliposomal K+ or Na+ showed that Na+ permeates through the transporter, yet KtrAB favors K+ over Na+. In addition, the cytosolic ring regulates the activity of the transmembrane pore, however, it does not render selectivity towards ions as there are no selectivity differences between the KtrAB complex, either with ADP or ATP, and KtrB alone. In conclusion, our data shows that KtrA regulates the activity of KtrB and raises new ideas about the mechanism of activation by RCK domains in the superfamily. Current investigation attempts to characterize the molecular basis of activation.

The lipocalin apolipoprotein D (ApoD) is associated in human plasma with lecithin-cholesterol acyl transferase. It has also been found in high concentration in the fluid of gross cystic disease of the mammary gland. Using protein fluorescence quenching, it is shown that ApoD binds arachidonic acid ( K a of 1.6 × 10 8 M −1 ) and as previously known progesterone ( K a of 2.5 × 10 6 M −1 ), but neither cholesterol nor any of the other prostanoid molecules examined had measurable affinity. This specific binding of arachidonate, also observable directly, suggests a role for ApoD in the mobilisation of Arachidonic acid, and hence prostaglandin synthesis.

The voltage-gated ether-a-go-go (EAG) potassium channel family plays an important role in cardiac and neuronal repolarization, tumor proliferation and hormone secretion. These channels are tetrameric, with six transmembrane helices per subunit and large N- and C-terminal cytoplasmic domains. The function of the cytoplasmic domains remains unclear, but it is thought that they are involved in channel regulation since they can be phosphorylated and can interact with kinases, integrins and calmodulin. In particular, the C-terminal domain has strong homology to cyclic nucleotide binding domains (CNBDs) but it has been demonstrated that EAG channel function is not affected by cyclic nucleotides and that the domain does not bind nucleotides, in vitro. I have determined the 2.2 A X-ray structure of one of these domains from the mouse EAG1 channel; the structure has the canonical CNBD fold but it shows a ligand pocket that has several differences relative to a cyclic nucleotide binding site, shedding some light into why these domains are not able to bind cyclic nucleotides. Here I present the biochemical characterization of this domain, a first step in the understanding the role of the C-terminal cytoplasmic domain in channel regulation.

Introduction Over the last decades, visceral leishmaniasis (VL) has spread to a number of metropolitan areas in Brazil. The aim was to determine the prevalence and the risk factors associated with Leishmania infantum infection among seronegative and molecular positive dogs in an area of active transmission in Southeast of Brazil. Methods A cross-sectional study was conducted with 1443 dogs, random selected. Molecular methods were performed using polymerase chain reaction (PCR-RFLP) and serology was conducted using ELISA. Mixed logistic regression model was used to evaluate the risk factors associated with L infantum infection. Principal findings Of the 1443 dogs examined, 230 (15.9%) were seropositive in ELISA assay, while PCR-RFLP revealed that 356 animals (24.7%) were positive and 1087 (75.3%) negative for L infantum DNA. To identify the risk factors associated with infection, only seronegative animals (n=1213) were evaluated, and of these 296 (24.4%) were PCR-positive and 917 (75.6%) PCR-negative for L infantum DNA. The risk factors associated with infection were family income < one minimum salary (OR.4.1; 95% CI 1.0 to 17.5), knowledge of the owner regarding the vector (OR.1.9; 95% CI 1.1 to 3.4), permanence of dog in the backyard (OR.2.3; 95% CI 1.2 to 4.4), and lack of examination for VL (OR.1.6; 95% CI.1.0 to 2.4). Conclusions PCR is more accurate than serology in the early detection of infection among asymptomatic dogs, and its application on a large scale for the surveillance of VL could minimise false-negative results. Identification of the risk factors associated with canine visceral leishmaniasis is important for the definition of strategies of disease control.

Heart failure (HF) is a dynamic clinical syndrome depending on multiple hemodynamic and neurohormonal factors. This syndrome concerns not only left ventricular systolic dysfunction but also left ventricular diastolic dysfunction and right ventricular dysfunction. Clinical features and therapeutic approaches are different for each of them. NYHA class IV is just one of the various prognostic factors of heart failure; consequently, severe heart failure is not synonymous of NYHA class IV.To study hospitalised patients with heart failure in NYHA class IV, in order to characterise the predominant dysfunction, and analyze evolution and mortality.A retrospective analysis of a prospective study.Hundred and eight hospitalised patients (1985-89). Patients with chronic obstructive pulmonary disease and acute myocardial infarction were excluded.Sixty nine patients: 29 female and 40 male, aged 18 to 81 years old (m = 59 +/- 15.5).Patients were clinically examined and had chest radiographs, electrocardiogram and M + 2D mode echocardiogram. Three groups were identified: Group I-patients with predominant left ventricular systolic dysfunction; Group II-patients with predominant left ventricular diastolic dysfunction; Group III-patients with predominant right ventricular dysfunction.41% of the patients had coronary artery disease; 44%, valvular heart disease; 11.8% dilated cardiomyopathy; 8.7% hypertrophic cardiomyopathy; 8.7% hypertensive cardiomyopathy. Fifty five percent of the patients were in group I and the major aetiology were coronary artery disease and valvular heart disease; 25% of the patients were in group II and the major aetiology were coronary artery disease and hypertrophic cardiomyopathy; 20% of the patients were in group III, all had valvular heart disease. The global mortality during the hospitalisation period was 15.9%, mostly group III (29%) and II (17%).Heart failure patients in NYHA class IV formed an heterogeneous group, requiring individualised therapeutic approaches. Left ventricular systolic dysfunction was the major pathophysiological mechanism, however, diastolic dysfunction and right ventricular dysfunction were frequent. Coronary artery disease presented as a frequent aetiology of heart failure, resulting in diastolic and/or systolic dysfunction. Valvular heart disease can be present as left ventricular systolic or diastolic dysfunction or as a right ventricular dysfunction, depending on the valvulopathy and the time of evolution. Echocardiography, in association with clinical features, has been very useful for each patient approach, allowing HF aetiology and pathophysiological mechanisms characterisation. The low mortality observed in this study was related certainly to the correction of precipitating factors, together with early etiologic diagnosis and pathophysiological characterisation, and adequate individualised treatment.

MlotiK1 is a cyclic nucleotide-dependent ion channel which contains an intracellular C-terminal cyclic nucleotide binding domain (CNB domain). We have used X-ray crystallography to determine several different structures of the MlotiK1 CNB domain structures in the bound and unbound state. In combination, the five MlotiK1 CNB domain structures provide a unique opportunity for analyzing, within a single protein, the structural differences between the apo and bound states and the structural variability within each state. With this analysis as a guide, we have probed the nucleotide selectivity and importance of specific residue side chains in ligand binding and channel activation. These data help to identify ligand-protein interactions that are important for ligand-dependence in this channel and more globally in the class of nucleotide-dependent proteins.

Thirteen (8.0%) of 162 blood donors from Uberaba-Minas Gerais State showed neutralizing antibodies to the vesiculovirus Piry. The previous residence in rural area (p less than 0.0001) and in the cities of the São Paulo State (p less than 0.05) were statistically more frequent in the Piry seropositive group.

In the course of a German-Portuguese research project, the possibilities of protein production from wine distillation wastewaters have been investigated. The method used is a single stage aeration system preceded by sedimentation. The process is characterized by good pollution removal efficiency and a high production of biosludge of high quality for animal feed stuff. The project can be regarded as promising.

Potassium ion flux across cellular membranes is mediated by a variety of protein channels and transporters. While ion channels provide pores through which select ions move down their electrical or chemical gradients at very high flux rates, ion transporters, on the other hand, show saturation for their substrates and may drive ions up their electrochemical gradient. Together, ion channels and transporters are critical components in the maintenance of cellular homeostasis, including osmolarity and pH as well as in electrical impulse propagation in higher organisms. We have previously demonstrated the RCK-domain octameric ring conformation for KtrAB potassium transporter. We focused on further characterizing this structural organization, obtaining high resolution atomic details by X-ray crystallography. We have therefore optimized overproduction and purification of full-length cytoplasmic (KtrA) and membranar (KtrB) components as well as the complex assembly. Ultimately, structural details will help understand conduction and regulation of potassium ion transport in KtrAB.

Members of the ether-a -go-go family are voltage-gated K channels possessing a C-terminal domain with homology to cyclic nucleotide-gated channels (CNG/HCN) (1). Despite this similarity, these channels are not directly modulated by cyclic nucleotides (2,3). Recent X-ray crystal structures their cyclic nucleotide binding homology domains (CNBhD's) have a structure corresponding to the binding pocket of HCN channels occupied with tyrosine (Y) and leucine (L) side chains on a re-entrant beta strand (4,5). Thus, the structure appears self-liganded. Functional analysis using double-alanine mutagenesis indicates these residues are involved in gating in both zebrafish ELK and human EAG1 (4,5). To further investigate whether the EAG1 CNBhD is in the “liganded” or “unliganded” state, we evaluated kinetics of activation using two-electrode voltage clamp and excised macropatch recordings from oocytes. Ten-to-90% rise time of activation increased more than 3-fold in Y699A/L701A (“unliganded”) mutants. Single alanine mutations failed to slow activation, suggesting the side chain at either position is sufficient to mediate the liganded conformation. Single or double aspartate substitutions phenocopied the slow activation of double-alanine mutant. Modifying a binding-pocket cysteine with MTSET, or deleting the ligand-containing loop, similarly slowed activation. Despite removing the intrinsic ligand, cyclic nucleotides failed to modulate the mutant channel in excised macropatches or bind to the mutant protein domain in fluorescent studies in vitro. Like activation, deactivation is also slowed. These findings support the conclusion that the double-alanine mutation creates an unliganded binding pocket, and suggest the role of the CNBhD is to reduce the energy barrier between closed and open states of the channel.1. Warmke et al. (1994) PNAS 91:34382. Robertson et al. (1996) Neuropharmacology 35:8413. Brelidze et al. (2009) JBC 284:279894. Brelidze et al. (2012) Nature 481:5305. Marques-Carvalho et al. (2012) JMB 423:34