A key unresolved question regarding the basic function of voltage-gated ion channels is how movement of the voltage sensor is coupled to channel opening. We previously proposed that the S4-S5 linker couples voltage sensor movement to the S6 domain in the human ether-a'-go-go-related gene (hERG) K ؉ channel. The recently solved crystal structure of the voltage-gated Kv1.2 channel reveals that the S4-S5 linker is the structural link between the voltage sensing and pore domains. In this study, we used chimeras constructed from hERG and ether-a'-go-go (EAG) channels to identify interactions between residues in the S4-S5 linker and S6 domain that were critical for stabilizing the channel in a closed state. To verify the spatial proximity of these regions, we introduced cysteines in the S4-S5 linker and at the C-terminal end of the S6 domain and then probed for the effect of oxidation. The D540C-L666C channel current decreased in an oxidizing environment in a state-dependent manner consistent with formation of a disulfide bond that locked the channel in a closed state. Disulfide bond formation also restricted movement of the voltage sensor, as measured by gating currents. Taken together, these data confirm that the S4-S5 linker directly couples voltage sensor movement to the activation gate. Moreover, rather than functioning simply as a mechanical lever, these findings imply that specific interactions between the S4-S5 linker and the activation gate stabilize the closed channel conformation.A fundamental property of all voltage-gated ion channels is the ability to open or close in response to changes in membrane potential. In voltage-gated K ϩ channels, the transmembrane domains are partitioned into distinct functional modules: a voltage-sensing module (S1-S4) and an ion-conducting module (S5-S6). Changes in the transmembrane electrical field exert a force on the highly charged S4 domain to initiate a process that culminates in the opening of the activation gate (1). The activation gate is formed by crisscrossing of the C-terminal portions of the S6 ␣-helices to form a narrow aperture near the cytoplasmic interface (2). Channel opening is proposed to involve splaying of the S6 helices at a conserved glycine, thereby widening the aperture to allow passage of ions (3, 4). Although substantial progress has been made in defining the structural basis of the voltage sensor and activation gate, the mechanism of coupling voltage sensing to channel opening, "electromechanical coupling" (5), remains poorly defined. Electromechanical coupling may involve global rearrangements between large domains within the channel complex, for example, outward movement of S4 coupled to rearrangements in S5 that are transmitted to S6. Alternatively, the coupling mechanism might involve discreet interactions between specific residues in more localized regions. The intracellular S4-S5 linker is ideally suited to function as an electromechanical coupler given that it is physically tethered to the S4 and, as such, could function to transduce voltag...
Although chloroquine remains an important therapeutic agent for treatment of malaria in many parts of the world, its safety margin is very narrow. Chloroquine inhibits the cardiac inward rectifier K ؉ current IK1 and can induce lethal ventricular arrhythmias. In this study, we characterized the biophysical and molecular basis of chloroquine block of Kir2.1 channels that underlie cardiac I K1. The voltage-and K ؉ -dependence of chloroquine block implied that the binding site was located within the ion-conduction pathway. Site-directed mutagenesis revealed the location of the chloroquine-binding site within the cytoplasmic pore domain rather than within the transmembrane pore. Molecular modeling suggested that chloroquine blocks Kir2.1 channels by plugging the cytoplasmic conduction pathway, stabilized by negatively charged and aromatic amino acids within a central pocket. Unlike most ionchannel blockers, chloroquine does not bind within the transmembrane pore and thus can reach its binding site, even while polyamines remain deeper within the channel vestibule. These findings explain how a relatively low-affinity blocker like chloroquine can effectively block I K1 even in the presence of high-affinity endogenous blockers. Moreover, our findings provide the structural framework for the design of safer, alternative compounds that are devoid of Kir2.1-blocking properties.IK1 ͉ ion channel ͉ KCNJ2 ͉ malaria ͉ polyamines
Objective To evaluate quantitative measures of saccades as possible biomarkers in early stages of Parkinson disease (PD) and in a population at-risk for PD. Methods The study sample (n = 68) included mildly to moderately affected PD patients, their unaffected siblings, and control individuals. All participants completed a clinical evaluation by a movement disorder neurologist. Genotyping of the G2019S mutation in the LRRK2 gene was performed in the PD patients and their unaffected siblings. A high resolution, video-based eye tracking system was employed to record eye positions during a battery of visually guided, anti-saccadic (AS), and two memory-guided (MG) tasks. Saccade measures (latency, velocity, gain, error rate, and multiple step pattern) were quantified. Results PD patients and a subgroup of their unaffected siblings had an abnormally high incidence of multiple step patterns (MSP) and reduced gain of saccades as compared with controls. The abnormalities were most pronounced in the more challenging version of the MG task. For this task, the MSP measure demonstrated good sensitivity (87%) and excellent specificity (96%) in the ability to discriminate PD patients from controls. PD patients and their siblings also made more errors in the AS task. Conclusions Abnormalities in eye movement measures appear to be sensitive and specific measures in PD patients as well as a subset of those at-risk for PD. The inclusion of quantitative laboratory testing of saccadic movements may increase the sensitivity of the neurological examination to identify individuals who are at greater risk for PD.
Objective To examine rates of decline in individuals at risk for Huntington disease (HD). Methods 106 individuals at risk for HD completed a battery of neurocognitive, psychomotor and oculomotor tasks at two visits, approximately 2.5 years apart. Participants were classified as: (1) without the CAG expansion (normal controls, NC; n=68) or (2) with the CAG expansion (CAG+; n=38). The CAG+ group was further subdivided into those near to (near; n=19) or far from (far; n=19) their estimated age of onset. Longitudinal performance in the CAG+ group was evaluated with a repeated measures model with two main effects (time to onset, visit) and their interaction. Analysis of covariance was employed to detect differences in longitudinal performance in the three groups (NC, near and far). Results In the CAG+, the interaction term was significant (p≤0.02) for four measures (movement time, alternate button tapping, variability of latency for a memory guided task and percentage of errors for a more complex memory guided task), suggesting the rate of decline was more rapid as subjects approached onset. Longitudinal progression in the three groups differed for several variables (p<0.05). In most, the near group had significantly faster progression than NC; however, comparisons of the NC and far groups were less consistent. Conclusions Different patterns of progression were observed during the prediagnostic period. For some measures, CAG+ subjects closer to estimated onset showed a more rapid decline while for other measures the CAG+ group had a constant rate of decline throughout the prediagnostic period that was more rapid than in NC.
HERG1 K+ channels are critical for modulating the duration of the cardiac action potential. The role of hERG1 channels in maintaining electrical stability in the heart derives from their unusual gating properties: slow activation and fast inactivation. HERG1 channel inactivation is intrinsically voltage sensitive and is not coupled to activation in the same way as in the Shaker family of K+ channels. We recently proposed that the S4 transmembrane domain functions as the primary voltage sensor for hERG1 activation and inactivation and that distinct regions of S4 contribute to each gating process. In this study, we tested the hypothesis that S4 rearrangements underlying activation and inactivation gating may be associated with distinct cooperative interactions between a key residue in the S4 domain (R531) and acidic residues in neighboring regions (S1 – S3 domains) of the voltage sensing module. Using double-mutant cycle analysis, we found that R531 was energetically coupled to all acidic residues in S1-S3 during activation, but was coupled only to acidic residues near the extracellular portion of S2 and S3 (D456, D460 and D509) during inactivation. We propose that hERG1 activation involves a cooperative conformational change involving the entire voltage sensing module, while inactivation may involve a more limited interaction between R531 and D456, D460 and D509.
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