Jérôme J. Lacroix

Control of a final gating charge transition by a hydrophobic residue in the S2 segment of a K+ channel voltage sensor

It is now well established that the voltage-sensor domains present in voltage-gated ion channels and some phosphatases operate by transferring several charged residues (gating charges), mainly arginines located in the S4 segment, across the electric field. The conserved phenylalanine F290 located in the S2 segment of the Shaker K channel is an aromatic residue thought to interact with all the four gating arginines carried by the S4 segment and control their transfer [Tao X, et al. (2010) Science 328:67–73]. In this paper we study the possible interaction of the gating charges with this residue by directly detecting their movement with gating current measurements in 12 F290 mutants. Most mutations do not significantly alter the first approximately 80–90% of the gating charge transfer nor the kinetics of the gating currents during activation. The effects of the F290 mutants are (i) the modification of a final activation transition accounting for approximately 10–20% of the total charge, similar to the effect of the ILT mutant [Ledwell JL, et al. (1999) J Gen Physiol 113:389–414] and (ii) the modification of the kinetics of the gating charge movement during deactivation. These effects are well correlated with the hydrophobicity of the substituted residue, showing that a hydrophobic residue at position 290 controls the energy barrier of the final gating transition. Our results suggest that F290 controls the transfer of R371, the fourth gating charge, during gating while not affecting the movement of the other three gating arginines.

Controlling the activity of a phosphatase and tensin homolog (PTEN) by membrane potential

The recently discovered voltage-sensitive phosphatases (VSPs) hydrolyze phosphoinositides upon depolarization of the membrane potential, thus representing a novel principle for the transduction of electrical activity into biochemical signals. Here, we demonstrate the possibility to confer voltage sensitivity to cytosolic enzymes. By fusing the tumor suppressor PTEN to the voltage sensor of the prototypic VSP from Ciona intestinalis, Ci-VSP, we generated chimeric proteins that are voltage-sensitive and display PTEN-like enzymatic activity in a strictly depolarization-dependent manner in vivo. Functional coupling of the exogenous enzymatic activity to the voltage sensor is mediated by a phospholipid-binding motif at the interface between voltage sensor and catalytic domains. Our findings reveal that the main domains of VSPs and related phosphoinositide phosphatases are intrinsically modular and define structural requirements for coupling of enzymatic activity to a voltage sensor domain. A key feature of this prototype of novel engineered voltage-sensitive enzymes, termed Ci-VSPTEN, is the novel ability to switch enzymatic activity of PTEN rapidly and reversibly. We demonstrate that experimental control of Ci-VSPTEN can be obtained either by electrophysiological techniques or more general techniques, using potassium-induced depolarization of intact cells. Thus, Ci-VSPTEN provides a novel approach for studying the complex mechanism of activation, cellular control, and pharmacology of this important tumor suppressor. Moreover, by inducing temporally precise perturbation of phosphoinositide concentrations, Ci-VSPTEN will be useful for probing the role and specificity of these messengers in many cellular processes and to analyze the timing of phosphoinositide signaling.

Intermediate state trapping of a voltage sensor.

Voltage sensor domains (VSDs) regulate ion channels and enzymes by undergoing conformational changes depending on membrane electrical signals. The molecular mechanisms underlying the VSD transitions are not fully understood. Here, we show that some mutations of I241 in the S1 segment of the Shaker Kv channel positively shift the voltage dependence of the VSD movement and alter the functional coupling between VSD and pore domains. Among the I241 mutants, I241W immobilized the VSD movement during activation and deactivation, approximately halfway between the resting and active states, and drastically shifted the voltage activation of the ionic conductance. This phenotype, which is consistent with a stabilization of an intermediate VSD conformation by the I241W mutation, was diminished by the charge-conserving R2K mutation but not by the charge-neutralizing R2Q mutation. Interestingly, most of these effects were reproduced by the F244W mutation located one helical turn above I241. Electrophysiology recordings using nonnatural indole derivatives ruled out the involvement of cation-Π interactions for the effects of the Trp inserted at positions I241 and F244 on the channel’s conductance, but showed that the indole nitrogen was important for the I241W phenotype. Insight into the molecular mechanisms responsible for the stabilization of the intermediate state were investigated by creating in silico the mutations I241W, I241W/R2K, and F244W in intermediate conformations obtained from a computational VSD transition pathway determined using the string method. The experimental results and computational analysis suggest that the phenotype of I241W may originate in the formation of a hydrogen bond between the indole nitrogen atom and the backbone carbonyl of R2. This work provides new information on intermediate states in voltage-gated ion channels with an approach that produces minimum chemical perturbation.

Molecular mechanism for depolarization-induced modulation of Kv channel closure

Voltage-dependent potassium (Kv) channels provide the repolarizing power that shapes the action potential duration and helps control the firing frequency of neurons. The K+ permeation through the channel pore is controlled by an intracellularly located bundle-crossing (BC) gate that communicates with the voltage-sensing domains (VSDs). During prolonged membrane depolarizations, most Kv channels display C-type inactivation that halts K+ conduction through constriction of the K+ selectivity filter. Besides triggering C-type inactivation, we show that in Shaker and Kv1.2 channels (expressed in Xenopus laevis oocytes), prolonged membrane depolarizations also slow down the kinetics of VSD deactivation and BC gate closure during the subsequent membrane repolarization. Measurements of deactivating gating currents (reporting VSD movement) and ionic currents (BC gate status) showed that the kinetics of both slowed down in two distinct phases with increasing duration of the depolarizing prepulse. The biphasic slowing in VSD deactivation and BC gate closure was strongly correlated in time and magnitude. Simultaneous recordings of ionic currents and fluorescence from a probe tracking VSD movement in Shaker directly demonstrated that both processes were synchronized. Whereas the first slowing originates from a stabilization imposed by BC gate opening, the subsequent slowing reflects the rearrangement of the VSD toward its relaxed state (relaxation). The VSD relaxation was observed in the Ciona intestinalis voltage-sensitive phosphatase and in its isolated VSD. Collectively, our results show that the VSD relaxation is not kinetically related to C-type inactivation and is an intrinsic property of the VSD. We propose VSD relaxation as a general mechanism for depolarization-induced slowing of BC gate closure that may enable Kv1.2 channels to modulate the firing frequency of neurons based on the depolarization history.

Moving Gating Charges through the Gating Pore in a Kv Channel Voltage-Sensor

Significance Voltage sensors are integral membrane protein domains that regulate ion channels and enzymes by transporting electrically charged residues across a narrow constriction that focuses the membrane electrical field. Here, we investigated how this constriction, also called the “gating pore,” controls this transport by studying the effects of a large number of point mutations. Our analysis indicates the presence of nonambiguous statistical correlations between specific amino acid lateral-chain physicochemical properties (size, hydrophobicity) and specific functional features of the voltage sensor (voltage sensitivity and transport kinetics). This study allowed us to propose engineering-like mechanisms by which gating pore residues control the voltage sensor operation. Voltage sensor domains (VSDs) regulate ion channels and enzymes by transporting electrically charged residues across a hydrophobic VSD constriction called the gating pore or hydrophobic plug. How the gating pore controls the gating charge movement presently remains debated. Here, using saturation mutagenesis and detailed analysis of gating currents from gating pore mutations in the Shaker Kv channel, we identified statistically highly significant correlations between VSD function and physicochemical properties of gating pore residues. A necessary small residue at position S240 in S1 creates a “steric gap” that enables an intracellular access pathway for the transport of the S4 Arg residues. In addition, the stabilization of the depolarized VSD conformation, a hallmark for most Kv channels, requires large side chains at positions F290 in S2 and F244 in S1 acting as “molecular clamps,” and a hydrophobic side chain at position I237 in S1 acting as a local intracellular hydrophobic barrier. Finally, both size and hydrophobicity of I287 are important to control the main VSD energy barrier underlying transitions between resting and active states. Taken together, our study emphasizes the contribution of several gating pore residues to catalyze the gating charge transfer. This work paves the way toward understanding physicochemical principles underlying conformational dynamics in voltage sensors.

Tuning the Voltage-Sensor Motion with a Single Residue

The Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) represents the first discovered member of enzymes regulated by a voltage-sensor domain (VSD) related to the VSD found in voltage-gated ion channels. Although the VSD operation in Ci-VSP exhibits original voltage dependence and kinetics compared to ion channels, it has been poorly investigated. Here, we show that the kinetics and voltage dependence of VSD movement in Ci-VSP can be tuned over 2 orders of magnitude and shifted over 120 mV, respectively, by the size of a conserved isoleucine (I126) in the S1 segment, thus indicating the importance of this residue in Ci-VSP activation. Mutations of the conserved Phe in the S2 segment (F161) do not significantly perturb the voltage dependence of the VSD movement, suggesting a unique voltage sensing mechanism in Ci-VSP.

Kv3.1 uses a timely resurgent K+ current to secure action potential repolarization

High-frequency action potential (AP) transmission is essential for rapid information processing in the central nervous system. Voltage-dependent Kv3 channels play an important role in this process thanks to their high activation threshold and fast closure kinetics, which reduce the neurons refractory period. However, premature Kv3 channel closure leads to incomplete membrane repolarization, preventing sustainable AP propagation. Here, we demonstrate that Kv3.1b channels solve this problem by producing resurgent K+ currents during repolarization, thus ensuring enough repolarizing power to terminate each AP. Unlike previously described resurgent Na+ and K+ currents, Kv3.1bs resurgent current does not originate from recovery of channel block or inactivation but results from a unique combination of steep voltage-dependent gating kinetics and ultra-fast voltage-sensor relaxation. These distinct properties are readily transferrable onto an orthologue Kv channel by transplanting the voltage-sensors S3–S4 loop, providing molecular insights into the mechanism by which Kv3 channels contribute to high-frequency AP transmission.

Probing α-310 Transitions in a Voltage-Sensing S4 Helix

The S4 helix of voltage sensor domains (VSDs) transfers its gating charges across the membrane electrical field in response to changes of the membrane potential. Recent studies suggest that this process may occur via the helical conversion of the entire S4 between α and 310 conformations. Here, using LRET and FRET, we tested this hypothesis by measuring dynamic changes in the transmembrane length of S4 from engineered VSDs expressed in Xenopus oocytes. Our results suggest that the native S4 from the Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) does not exhibit extended and long-lived 310 conformations and remains mostly α-helical. Although the S4 of NavAb displays a fully extended 310 conformation in x-ray structures, its transplantation in the Ci-VSP VSD scaffold yielded similar results as the native Ci-VSP S4. Taken together, our study does not support the presence of long-lived extended α-to-310 helical conversions of the S4 in Ci-VSP associated with voltage activation.

Development of a PET radioligand for potassium channels to image CNS demyelination

Central nervous system (CNS) demyelination represents the pathological hallmark of multiple sclerosis (MS) and contributes to other neurological conditions. Quantitative and specific imaging of demyelination would thus provide critical clinical insight. Here, we investigated the possibility of targeting axonal potassium channels to image demyelination by positron emission tomography (PET). These channels, which normally reside beneath the myelin sheath, become exposed upon demyelination and are the target of the MS drug, 4-aminopyridine (4-AP). We demonstrate using autoradiography that 4-AP has higher binding in non-myelinated and demyelinated versus well-myelinated CNS regions, and describe a fluorine-containing derivative, 3-F-4-AP, that has similar pharmacological properties and can be labeled with 18F for PET imaging. Additionally, we demonstrate that [18F]3-F-4-AP can be used to detect demyelination in rodents by PET. Further evaluation in Rhesus macaques shows higher binding in non-myelinated versus myelinated areas and excellent properties for brain imaging. Together, these data indicate that [18F]3-F-4-AP may be a valuable PET tracer for detecting CNS demyelination noninvasively.

Probing S4 Length Changes during Gating with LRET

Voltage-activated proteins containing a Voltage Sensor Domain (VSD) respond to changes in the membrane potential by transferring across the electric field several positively charged residues (gating charges) located in the fourth transmembrane segment (S4). Even though the mechanistic details of gating charge translocation and S4 re-arrangement are presently unclear, a prevailing hypothesis suggests that the S4 segment adopts a 310 helix conformation during gating, thus aligning the gating charges and facilitating their transport across the membrane electric field. If a whole typical S4 segment were to change its conformation from an α-helix to a 310 helix, its length would stretch by about 8 A. Here we tested the existence of such transition by measuring the length of the S4 segment during gating using the LRET technique. We used the VSD domain of the Ciona intestinalis Voltage-Sensitive Phosphatase (CiVSP), truncated from its phosphatase and phospholipid-binding domains, to genetically encode a lanthanide (Tb3+)-binding-tag at the extracellular end of S4 and the red fluorescent protein mCherry at its intracellular end. We expressed the protein in Xenopus oocytes from which we recorded gating currents using the cut-open and two-electrode voltage-clamp techniques. The distance-dependent efficiency of energy transfer between the two probes was measured at steady-state voltages and also during voltage pulse protocols of varying amplitudes and durations. Our technique could only detect distance changes larger than 2.5 A. We found that in our CiVSP construct, the results are not consistent with the expected length change if the whole S4 were converted (and remained converted) from a 310 helix to an α-helix (or vice-versa) when the membrane potential is changed from −100 to +80 mV. Supported by NIH GM68044-07 and GM30376-30S1.