Limited Impact of Cannabidiol on Health-related Quality of Life of People With Long-term Controlled HIV: A Double-blind, Randomized, Controlled Trial.

Background: People with HIV (PWH) with undetectable HIV viral load still have an impaired health-related quality of life (HRQoL). Cannabidiol (CBD) is a nonintoxicating cannabis-derived cannabinoid that holds promise for the treatment of many ailments. In the present study, we tested whether oral CBD-rich medication could significantly improve PWH's HRQoL. Methods: Eighty participants with undetectable HIV viral load were randomized to either a placebo or full-spectrum CBD (1 mg/kg twice a day) arm for 12 weeks plus a 4-week follow-up period. HRQoL was assessed at baseline, week 12, and week 16 using the 36-Item Short Form Health Survey questionnaire (SF-36). Primary outcomes were physical and mental component summary scores; secondary outcomes were the 8 SF-36 subscale scores. Treatment effects on outcomes were estimated using generalized estimating equations. Results: We found no effect of CBD intake on the summary score for either component. However, CBD intake was associated with a higher physical functioning score at week 12 only (regression coefficient [95% confidence interval], 7.72 [0.55-14.89]; P = .035). No significant main effect of CBD intake on the other HRQoL subscale scores was observed. Furthermore, there was no difference in self-reported adverse effects between the 2 arms. Conclusions: Twice-daily CBD full-spectrum oil at 1 mg/kg had no major effect on virologically suppressed PWH's HRQoL but had a positive effect on physical functioning. Further randomized controlled trials including PWH with lower baseline HRQoL are needed to confirm this finding.

Cognitive behavioural therapy for insomnia (CBTi) as a treatment for tinnitus-related insomnia: a randomised controlled trial.

Insomnia is a significant difficulty and is reported by large proportion of people with tinnitus. Although cognitive behavioural therapy for insomnia (CBTi) might be an effective treatment, no controlled studies had been conducted to date. This randomised controlled trial evaluated the benefits of CBTi on a sample of 102 people with tinnitus-related insomnia. Participants were randomised to 1) CBTi, 2) Audiology-Based Care (ABC) or 3) Sleep Support Group (SSG). Primary outcomes included insomnia, sleep efficiency and total sleep time. Secondary outcomes measured sleep onset latency, sleep quality, tinnitus distress, psychological distress, functioning and quality of life. CBTi was superior at reducing insomnia and increasing sleep efficiency compared to ABC post-intervention and at 6-month follow-up. ABC was superior at reducing insomnia and increasing sleep efficiency compared to SSG. Both CBTi and ABC reported increased total sleep time compared to SSG at 6-month follow. More than 80% of participants in the CBTi group reported clinically meaningful improvements compared to 47% in ABC and 20% for those receiving social support. CBTi was more effective in reducing tinnitus distress and improving sleep quality, functioning and some aspects of mental health. CBTi and ABC offer effective treatments for tinnitus-related sleep disorder but CBTi offers a sizeable benefit.

Cognitive Behavioral Therapy for Patients with Trigeminal Neuralgia: A Feasibility Study.

AIMS: To test the feasibility and acceptability of a customized six-session cognitive behavioral therapy (CBT) group intervention for adults with recurrent trigeminal neuralgia (TN). METHODS: Fifteen participants with TN were recruited from a specialist facial pain unit in London, United Kingdom. The effects of the group intervention were evaluated using validated self-report measures, which the participants completed before and after the intervention and at 1-month and 9-month follow-ups. A semi-structured interview was also used at the 1-year follow-up to gather qualitative feedback of the group intervention. RESULTS: Participants reported an increase in confidence in managing everyday tasks in the presence of TN symptoms, a reduction in negative beliefs about pain, and an increase in engagement in meaningful activity. All patients completed the group intervention (100% retention rate). Qualitative feedback highlighted that the group CBT intervention was helpful, and no participants reported a worsening of mood or experience as a result of the intervention. CONCLUSION: The trends for improvement in several domains, plus the positive experiences of the participants, suggest that a CBT management program is acceptable and feasible for this population and should be further developed and implemented on a larger scale to determine its clinical efficacy.

Using wearable monitors to assess daily walking limitations induced by ischemic pain in peripheral artery disease.

PURPOSE: To develop, assess the feasibility of, and determine the clinical validity of an event-based analysis method using wearable monitors to quantify walking pain manifestations (WPMs) and stops induced by walking pain (SIWPs) during daily life walking in people with peripheral artery disease (PAD). METHODS: The following two conditions were studied: a standardized outdoor walking session (OWS) and a seven-day free-living measurement (FLM) period. The PAD participants (n = 23) wore an accelerometer and a watch. They were asked to press the event marker button on the watch to indicate events related to WPMs and SIWPs. To assess the clinical validity of the method, the computed pain-free walking time (PFWT) and maximal walking time (MWT) were compared with the PFWT and MWT assessed using standard treadmill walking protocols, respectively. RESULTS: Following OWSs, the PFWT[OWS] and MWT[OWS] were significantly correlated with the PFWT[Strandness] (r = .955, P < .001) and MWT[Strandness] (r = .821, P < .001), respectively. During the FLM, PAD participants experienced only 2 WPMs/day and 1 SIWP/day, although severely limited on the treadmill and during the OWS. The average WPMs/day were moderately correlated with the PFWT[Strandness] (r = -.54, P = .016). The PFWT[FLM] was on average 12 times longer than the PFWT[Strandness] . Interestingly, the intensity of the walking bouts as assessed by the accelerometer counts during the FLM was significantly lower than that during the OWS (45 ± 15 vs 66 ± 20 counts/s, P < .001). CONCLUSION: This new method offers opportunities for studies investigating the experience of living with PAD and the assessment of daily life walking capacity for both diagnostic and therapeutic purposes.

Investigating CFTR and KCa3.1 Protein/Protein Interactions.

In epithelia, Cl- channels play a prominent role in fluid and electrolyte transport. Of particular importance is the cAMP-dependent cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) with mutations of the CFTR encoding gene causing cystic fibrosis. The bulk transepithelial transport of Cl- ions and electrolytes needs however to be coupled to an increase in K+ conductance in order to recycle K+ and maintain an electrical driving force for anion exit across the apical membrane. In several epithelia, this K+ efflux is ensured by K+ channels, including KCa3.1, which is expressed at both the apical and basolateral membranes. We show here for the first time that CFTR and KCa3.1 can physically interact. We first performed a two-hybrid screen to identify which KCa3.1 cytosolic domains might mediate an interaction with CFTR. Our results showed that both the N-terminal fragment M1-M40 of KCa3.1 and part of the KCa3.1 calmodulin binding domain (residues L345-A400) interact with the NBD2 segment (G1237-Y1420) and C- region of CFTR (residues T1387-L1480), respectively. An association of CFTR and F508del-CFTR with KCa3.1 was further confirmed in co-immunoprecipitation experiments demonstrating the formation of immunoprecipitable CFTR/KCa3.1 complexes in CFBE cells. Co-expression of KCa3.1 and CFTR in HEK cells did not impact CFTR expression at the cell surface, and KCa3.1 trafficking appeared independent of CFTR stimulation. Finally, evidence is presented through cross-correlation spectroscopy measurements that KCa3.1 and CFTR colocalize at the plasma membrane and that KCa3.1 channels tend to aggregate consequent to an enhanced interaction with CFTR channels at the plasma membrane following an increase in intracellular Ca2+ concentration. Altogether, these results suggest 1) that the physical interaction KCa3.1/CFTR can occur early during the biogenesis of both proteins and 2) that KCa3.1 and CFTR form a dynamic complex, the formation of which depends on internal Ca2+.

Aromatic-aromatic interactions between residues in KCa3.1 pore helix and S5 transmembrane segment control the channel gating process.

The Ca(2+)-activated potassium channel KCa3.1 is emerging as a therapeutic target for a large variety of health disorders. One distinguishing feature of KCa3.1 is that the channel open probability at saturating Ca(2+) concentrations (Pomax) is low, typically 0.1-0.2 for KCa3.1 wild type. This observation argues for the binding of Ca(2+) to the calmodulin (CaM)-KCa3.1 complex, promoting the formation of a preopen closed-state configuration leading to channel opening. We have previously shown that the KCa3.1 active gate is most likely located at the level of the selectivity filter. As Ca(2+)-dependent gating of KCa3.1 originates from the binding of Ca(2+) to CaM in the C terminus, the hypothesis of a gate located at the level of the selectivity filter requires that the conformational change initiated in the C terminus be transmitted to the S5 and S6 transmembrane helices, with a resulting effect on the channel pore helix directly connected to the selectivity filter. A study was thus undertaken to determine to what extent the interactions between the channel pore helix with the S5 and S6 transmembrane segments contribute to KCa3.1 gating. Molecular dynamics simulations first revealed that the largest contact area between the pore helix and the S5 plus S6 transmembrane helices involves residue F248 at the C-terminal end of the pore helix. Unitary current recordings next confirmed that modulating aromatic-aromatic interactions between F248 and W216 of the S5 transmembrane helical segment and/or perturbing the interactions between F248 and residues in S6 surrounding the glycine hinge G274 cause important changes in Pomax. This work thus provides the first evidence for a key contribution of the pore helix in setting Pomax by stabilizing the channel closed configuration through aromatic-aromatic interactions involving F248 of the pore helix. We propose that the interface pore helix/S5 constitutes a promising site for designing KCa3.1 potentiators.

Contribution of the KCa3.1 channel-calmodulin interactions to the regulation of the KCa3.1 gating process.

The Ca(2+)-activated potassium channel of intermediate conductance, KCa3.1, is now emerging as a therapeutic target for a large variety of health disorders. The Ca(2+) sensitivity of KCa3.1 is conferred by the Ca(2+)-binding protein calmodulin (CaM), with the CaM C-lobe constitutively bound to an intracellular domain of the channel C terminus. It was proposed on the basis of the crystal structure obtained for the C-terminal region of the rat KCa2.2 channel (rSK2) with CaM that the binding of Ca(2+) to the CaM N-lobe results in CaM interlocking the C-terminal regions of two adjacent KCa3.1 subunits, leading to the formation of a dimeric structure. A study was thus undertaken to identify residues of the CaM N-lobe-KCa3.1 complex that either contribute to the channel activation process or control the channel open probability at saturating Ca(2+) (Pomax). A structural homology model of the KCa3.1-CaM complex was first generated using as template the crystal structure of the C-terminal region of the rat KCa2.2 channel with CaM. This model was confirmed by cross-bridging residues R362 of KCa3.1 and K75 of CaM. Patch-clamp experiments were next performed, demonstrating that the solvation energy of the residue at position 367 in KCa3.1 is a key determinant to the channel Pomax and deactivation time toff. Mutations of residues M368 and Q364 predicted to form anchoring points for CaM binding to KCa3.1 had little impact on either toff or Pomax. Finally, our results show that channel activation depends on electrostatic interactions involving the charged residues R362 and E363, added to a nonpolar energy contribution coming from M368. We conclude that electrostatic interactions involving residues R362 and E363 and hydrophobic effects at M368 play a prominent role in KCa3.1 activation, whereas hydrophobic interactions at S367 are determinant to the stability of the CaM-KCa3.1 complex throughout gating.

Toward the rational design of constitutively active KCa3.1 mutant channels.

The Ca²+ activated potassium channel of intermediate conductance KCa3.1 is now emerging as a therapeutic target for a large variety of health disorders. KCa3.1 is a tetrameric membrane protein with each subunit formed of six transmembrane helices (S1-S6). Ca²+ sensitivity is conferred by the Ca²+ binding protein calmodulin (CaM), with the CaM C-lobe constitutively bound to an intracellular domain of the channel C-terminus, located proximal to the membrane and connected to the S6 transmembrane segment. Patch clamp single channel recordings have demonstrated that binding of Ca²+ to CaM allows the channel to transit dose dependently from a nonconducting to an ion-conducting configuration. Here we present a general strategy to generate KCa3.1 mutant channels that remain in an ion-conducting state in the absence of Ca²+. Our strategy is first based on the production of a 3D model of the channel pore region, followed by SCAM experiments to confirm that residues along each of the channel S6 transmembrane helix form the channel pore lumen as predicted. In a simple model, constitutive activity can be obtained by removing the steric hindrances inside the channel pore susceptible to prevent ion flow when the channel is in the closed configuration. Using charged MTS reagents and Ag+ ions as probes acting on Cys residues engineered in the pore lumen, we found that the S6 transmembrane helices of KCa3.1 cannot form a pore constriction tight enough to prevent ion flow for channels in the closed state. These observations ruled out experimental strategies where constitutive activity would be generated by producing a "leaky" closed channel. A more successful approach consisted however in perturbing the channel open/closed state equilibrium free energy. In particular, we found that substituting the hydrophobic residue V282 in S6 by hydrophilic amino acids could lock the channel in an open-like state, resulting in channels that were ion conducting in the absence of Ca²+.

Molecular determinants of the CaVbeta-induced plasma membrane targeting of the CaV1.2 channel.

Ca(V)beta subunits modulate cell surface expression and voltage-dependent gating of high voltage-activated (HVA) Ca(V)1 and Ca(V)2 alpha1 subunits. High affinity Ca(V)beta binding onto the so-called alpha interaction domain of the I-II linker of the Ca(V)alpha1 subunit is required for Ca(V)beta modulation of HVA channel gating. It has been suggested, however, that Ca(V)beta-mediated plasma membrane targeting could be uncoupled from Ca(V)beta-mediated modulation of channel gating. In addition to Ca(V)beta, Ca(V)alpha2delta and calmodulin have been proposed to play important roles in HVA channel targeting. Indeed we show that co-expression of Ca(V)alpha2delta caused a 5-fold stimulation of the whole cell currents measured with Ca(V)1.2 and Ca(V)beta3. To gauge the synergetic role of auxiliary subunits in the steady-state plasma membrane expression of Ca(V)1.2, extracellularly tagged Ca(V)1.2 proteins were quantified using fluorescence-activated cell sorting analysis. Co-expression of Ca(V)1.2 with either Ca(V)alpha2delta, calmodulin wild type, or apocalmodulin (alone or in combination) failed to promote the detection of fluorescently labeled Ca(V)1.2 subunits. In contrast, co-expression with Ca(V)beta3 stimulated plasma membrane expression of Ca(V)1.2 by a 10-fold factor. Mutations within the alpha interaction domain of Ca(V)1.2 or within the nucleotide kinase domain of Ca(V)beta3 disrupted the Ca(V)beta3-induced plasma membrane targeting of Ca(V)1.2. Altogether, these data support a model where high affinity binding of Ca(V)beta to the I-II linker of Ca(V)alpha1 largely accounts for Ca(V)beta-induced plasma membrane targeting of Ca(V)1.2.

Nitric oxide increases cardiac IK1 by nitrosylation of cysteine 76 of Kir2.1 channels.

RATIONALE: The cardiac inwardly rectifying K(+) current (I(K1)) plays a critical role in modulating excitability by setting the resting membrane potential and shaping phase 3 of the cardiac action potential. OBJECTIVE: This study aims to analyze the effects of nitric oxide (NO) on human atrial I(K1) and on Kir2.1 channels, the major isoform of inwardly rectifying channels present in the human heart. METHODS AND RESULTS: Currents were recorded in enzymatically isolated myocytes and in transiently transfected CHO cells, respectively. NO at myocardial physiological concentrations (25 to 500 nmol/L) increased inward and outward I(K1) and I(Kir2.1). These effects were accompanied by hyperpolarization of the resting membrane potential and a shortening of the duration of phase 3 of the human atrial action potential. The I(Kir2.1) increase was attributable to an increase in the open probability of the channel. Site-directed mutagenesis analysis demonstrated that NO effects were mediated by the selective S-nitrosylation of Kir2.1 Cys76 residue. Single ion monitoring experiments performed by liquid chromatography/tandem mass spectrometry suggested that the primary sequence that surrounds Cys76 determines its selective S-nitrosylation. Chronic atrial fibrillation, which produces a decrease in NO bioavailability, decreased the S-nitrosylation of Kir2.1 channels in human atrial samples as demonstrated by a biotin-switch assay, followed by Western blot. CONCLUSIONS: The results demonstrated that, under physiological conditions, NO regulates human cardiac I(K1) through a redox-related process.

Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells.

The vectorial transport of ions and water across epithelial cells depends to a large extent on the coordination of the apical and basolateral ion fluxes with energy supply. In this work we provide the first evidence for a regulation by the 5'-AMP-activated protein kinase (AMPK) of the calcium-activated potassium channel KCa3.1 expressed at the basolateral membrane of a large variety of epithelial cells. Inside-out patch-clamp experiments performed on human embryonic kidney (HEK) cells stably transfected with KCa3.1 first revealed a decrease in KCa3.1 activity following the internal addition of AMP at a fixed ATP concentration. This effect was dose dependent with half inhibition at 140 muM AMP in 1 mM ATP. Evidence for an interaction between the COOH-terminal region of KCa3.1 and the gamma1-subunit of AMPK was next obtained by two-hybrid screening and pull-down experiments. Our two-hybrid analysis confirmed in addition that the amino acids extending from Asp(380) to Ala(400) in COOH-terminal were essential for the interaction AMPK-gamma1/KCa3.1. Inside-out experiments on cells coexpressing KCa3.1 with the dominant negative AMPK-gamma1-R299G mutant showed a reduced sensitivity of KCa3.1 to AMP, arguing for a functional link between KCa3.1 and the gamma1-subunit of AMPK. More importantly, coimmunoprecipitation experiments carried out on bronchial epithelial NuLi cells provided direct evidence for the formation of a KCa3.1/AMPK-gamma1 complex at endogenous AMPK and KCa3.1 expression levels. Finally, treating NuLi monolayers with the membrane permeant AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) caused a significant decrease of the KCa3.1-mediated short-circuit currents, an effect reversible by coincubation with the AMPK inhibitor Compound C. These observations argue for a regulation of KCa3.1 by AMPK in a functional epithelium through protein/protein interactions involving the gamma1-subunit of AMPK.

Structural determinants of the closed KCa3.1 channel pore in relation to channel gating: results from a substituted cysteine accessibility analysis.

In this work we address the question of the KCa3.1 channel pore structure in the closed configuration in relation to the contribution of the C-terminal end of the S6 segments to the Ca(2+)-dependent gating process. Our results based on SCAM (substituted cysteine accessibility method) experiments first demonstrate that the S6 transmembrane segment of the open KCa3.1 channel contains two distinct functional domains delimited by V282 with MTSEA and MTSET binding leading to a total channel inhibition at positions V275, T278, and V282 and to a steep channel activation at positions A283 and A286. The rates of modification by MTSEA (diameter 4.6 A) of the 275C (central cavity) and 286C residues (S6 C-terminal end) for the closed channel configuration were found to differ by less than sevenfold, whereas experiments performed with the larger MTSET reagent (diameter 5.8 A) resulted in modification rates 10(3)-10(4) faster for cysteines at 286 compared with 275. Consistent with these results, the modification rates of the cavity lining 275C residue by MTSEA, Et-Hg(+), and Ag(+) appeared poorly state dependent, whereas modification rates by MTSET were 10(3) faster for the open than the closed configuration. A SCAM analysis of the channel inner vestibule in the closed state revealed in addition that cysteine residues at 286 were accessible to MTS reagents as large as MTS-PtrEA, a result supported by the observation that binding of MTSET to cysteines at positions 283 or 286 could neither sterically nor electrostatically block the access of MTSEA to the closed channel cavity (275C). It follows that the closed KCa3.1 structure can hardly be accountable by an inverted teepee-like structure as described for KcsA, but is better represented by a narrow passage centered at V282 (equivalent to V474 in Shaker) connecting the channel central cavity to the cytosolic medium. This passage would not be however restrictive to the diffusion of small reagents such as MTSEA, Et-Hg(+), and Ag(+), arguing against the C-terminal end of S6 forming an obstructive barrier to the diffusion of K(+) ions for the closed channel configuration.

Head repositioning accuracy in patients with whiplash-associated disorders.

STUDY DESIGN: Controlled study, measuring head repositioning error (HRE) using an electrogoniometric device. OBJECTIVE: To compare HRE in neutral position, axial rotation and complex postures of patients with whiplash-associated disorders (WAD) to that of control subjects. SUMMARY OF BACKGROUND DATA: The presence of kinesthetic alterations in patients with WAD is controversial. METHODS: In 26 control subjects and 29 patients with WAD (aged 22-74 years), head kinematics was sampled using a 3-dimensional electrogoniometer mounted using a harness and a helmet. All tasks were realized in seated position. The repositioning tasks included neutral repositioning after maximal flexion-extension, eyes open and blindfolded, repositioning at 50 degrees of axial rotation, and repositioning at 50 degrees of axial rotation combined to 20 degrees of ipsilateral bending. The flexion-extension, ipsilateral bending, and axial rotation components of HRE were considered. A multiple-way repeated-measures analysis of variance was used to compare tasks and groups. RESULTS: The WAD group displayed a reduced flexion-extension range (P = 1.9 x 10(-4)), and larger HRE during flexion-extension and repositioning tasks (P = 0.009) than controls. Neither group nor task affected maximal motion velocity. Neutral HRE of the flexion-extension component was larger in blindfolded condition (P = 0.03). Ipsilateral bending and axial rotation HRE components were smaller than the flexion-extension component (P = 7.1 x 10(-23)). For pure rotation repositioning, axial rotation HRE was significantly larger than flexion-extension and ipsilateral bending repositioning error (P = 3.0 x 10(-23)). Ipsilateral bending component of HRE was significantly larger combined tasks than for pure rotation tasks (P = 0.004). CONCLUSIONS: In patients with WAD, range of motion and head repositioning accuracy were reduced. However, the differences were small. Vision suppression and task type influenced HRE.

New insights on the voltage dependence of the KCa3.1 channel block by internal TBA.

We present in this work a structural model of the open IKCa (KCa3.1) channel derived by homology modeling from the MthK channel structure, and used this model to compute the transmembrane potential profile along the channel pore. This analysis showed that the selectivity filter and the region extending from the channel inner cavity to the internal medium should respectively account for 81% and 16% of the transmembrane potential difference. We found however that the voltage dependence of the IKCa block by the quaternary ammonium ion TBA applied internally is compatible with an apparent electrical distance delta of 0.49 +/- 0.02 (n = 6) for negative potentials. To reconcile this observation with the electrostatic potential profile predicted for the channel pore, we modeled the IKCa block by TBA assuming that the voltage dependence of the block is governed by both the difference in potential between the channel cavity and the internal medium, and the potential profile along the selectivity filter region through an effect on the filter ion occupancy states. The resulting model predicts that delta should be voltage dependent, being larger at negative than positive potentials. The model also indicates that raising the internal K+ concentration should decrease the value of delta measured at negative potentials independently of the external K+ concentration, whereas raising the external K+ concentration should minimally affect delta for concentrations >50 mM. All these predictions are born out by our current experimental results. Finally, we found that the substitutions V275C and V275A increased the voltage sensitivity of the TBA block, suggesting that TBA could move further into the pore, thus leading to stronger interactions between TBA and the ions in the selectivity filter. Globally, these results support a model whereby the voltage dependence of the TBA block in IKCa is mainly governed by the voltage dependence of the ion occupancy states of the selectivity filter.

Outer pore topology of the ECaC-TRPV5 channel by cysteine scan mutagenesis.

The substituted cysteine accessibility method (SCAM) was used to map the external vestibule and the pore region of the ECaC-TRPV5 calcium-selective channel. Cysteine residues were introduced at 44 positions from the end of S5 (Glu515) to the beginning of S6 (Ala560). Covalent modification by positively charged MTSET applied from the external medium significantly inhibited whole cell currents at 15/44 positions. Strongest inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET or MTSES suggesting that these residues were accessible from the external medium. In contrast, the pattern of covalent modification by MTSET for residues between Pro527 and Ile541 was compatible with the presence of a alpha-helix. The absence of modification by the negatively charged MTSES in that region suggests that the pore region has been optimized to favor the entrance of positively charged ions. Cysteine mutants at positions -1, 0, +1, +2 around Asp542 (high Ca2+ affinity site) were non-functional. Whole cell currents of cysteine mutants at +4 and +5 positions were however covalently inhibited by external MTSET and MTSES. Altogether, the pattern of covalent modification by MTS reagents globally supports a KcsA homology-based three-dimensional model whereby the external vestibule in ECaC-TRPV5 encompasses three structural domains consisting of a coiled structure (Glu515 to Tyr526) connected to a small helical segment of 15 amino acids (527PTALFSTFELFLT539) followed by two distinct coiled structures Ile540-Pro544 (selectivity filter) and Ala545-Ile557 before the beginning of S6.

Cysteine mutagenesis and computer modeling of the S6 region of an intermediate conductance IKCa channel.

Cysteine-scanning mutagenesis (SCAM) and computer-based modeling were used to investigate key structural features of the S6 transmembrane segment of the calcium-activated K(+) channel of intermediate conductance IKCa. Our SCAM results show that the interaction of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) with cysteines engineered at positions 275, 278, and 282 leads to current inhibition. This effect was state dependent as MTSET appeared less effective at inhibiting IKCa in the closed (zero Ca(2+) conditions) than open state configuration. Our results also indicate that the last four residues in S6, from A283 to A286, are entirely exposed to water in open IKCa channels, whereas MTSET can still reach the 283C and 286C residues with IKCa maintained in a closed state configuration. Notably, the internal application of MTSET or sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) caused a strong Ca(2+)-dependent stimulation of the A283C, V285C, and A286C currents. However, in contrast to the wild-type IKCa, the MTSET-stimulated A283C and A286C currents appeared to be TEA insensitive, indicating that the MTSET binding at positions 283 and 286 impaired the access of TEA to the channel pore. Three-dimensional structural data were next generated through homology modeling using the KcsA structure as template. In accordance with the SCAM results, the three-dimensional models predict that the V275, T278, and V282 residues should be lining the channel pore. However, the pore dimensions derived for the A283-A286 region cannot account for the MTSET effect on the closed A283C and A286 mutants. Our results suggest that the S6 domain extending from V275 to V282 possesses features corresponding to the inner cavity region of KcsA, and that the COOH terminus end of S6, from A283 to A286, is more flexible than predicted on the basis of the closed KcsA crystallographic structure alone. According to this model, closure by the gate should occur at a point located between the T278 and V282 residues.