Voltage sensors containing the charged S4 membrane segment display a gating charge vs. voltage (Q-V) curve that depends on the initial voltage. The voltage-dependent phosphatase (Ci-VSP), which does not have a conducting pore, shows the same phenomenon and the Q-V recorded with a depolarized initial voltage is more stable by at least 3RT. The leftward shift of the Q-V curve under prolonged depolarization was studied in the Ci-VSP by using electrophysiological and site-directed fluorescence measurements. The fluorescence shows two components: one that traces the time course of the charge movement between the resting and active states and a slower component that traces the transition between the active state and a more stable state we call the relaxed state. Temperature dependence shows a large negative enthalpic change when going from the active to the relaxed state that is almost compensated by a large negative entropic change. The Q-V curve midpoint measured for pulses that move the sensor between the resting and active states, but not long enough to evolve into the relaxed states, show a periodicity of 120°, indicating a 3 10 secondary structure of the S4 segment when determined under histidine scanning. We hypothesize that the S4 segment moves as a 3 10 helix between the resting and active states and that it converts to an ␣-helix when evolving into the relaxed state, which is most likely to be the state captured in the crystal structures.A number of membrane proteins respond to changes in the membrane electric field via an intrinsic voltage sensor in the protein structure (1). The classical examples, first described by Hodgkin and Huxley (2), are the voltage-dependent sodium and potassium conductances, which are crucial players in the generation and propagation of the nerve impulse. In the voltagegated ion channels that generate these conductances, the movement of the voltage sensor generates a transient current that has been traditionally called gating current, because in the original recordings it was correlated with the opening of the conduction pathway in Na channels (3, 4). Gating currents are transient because the sensing or gating charges are tethered to the protein and so their movement is restricted within the membrane electric field. Most of the moving charges that produce gating currents have been identified as the four most extracellular basic residues of the fourth transmembrane segment (S4) in voltage-gated channels (5, 6).At extremely negative and positive membrane potentials the gating charges are driven to extreme positions so that a plot of the transported charge as a function of the membrane potential (the Q-V curve) has a sigmoid shape that saturates at extreme potentials. The voltage dependence of this charge movement is an expression of the amount of charge involved and the number of states that the sensing charge populates when moving between the extreme positions. The steepness of the Q-V curve in going from one extreme position to the other increases by increasing the moving charge o...

SUMMARY The transduction of transmembrane electric fields into protein motion plays an essential role in the generation and propagation of cellular signals. Voltage-sensing domains (VSD) carry out these functions through reorientations of S4 helix with discrete gating charges. Here, crystal structures of the VSD from Ci-VSP were determined in both, active (Up) and resting (Down) conformations. The S4 undergoes a ~5 Å displacement along its main axis accompanied by a ~60o rotation, consistent with the helix-screw gating mechanism. This movement is stabilized by a change in countercharge partners in helices S1 and S3, generating an estimated net charge transfer of ~1 eo. Gating charges move relative to a “hydrophobic gasket” that electrically divides intra and extracellular compartments. EPR spectroscopy confirms the limited nature of S4 movement in a membrane environment. These results provide an explicit mechanism for voltage sensing and set the basis for electromechanical coupling in voltage-dependent cellular activities.

The Ciona intestinalis voltage sensor–containing phosphatase (Ci-VSP) shares high homology with the phosphatidylinositol phosphatase enzyme known as PTEN (phosphatase and tensin homologue deleted on chromosome 10). We have taken advantage of the similarity between these proteins to inquire about the coupling between the voltage sensing and the phosphatase domains in Ci-VSP. Recently, it was shown that four basic residues (R11, K13, R14, and R15) in PTEN are critical for its binding onto the membrane, required for its catalytic activity. Ci-VSP has three of the basic residues of PTEN. Here, we show that when R253 and R254 (which are the homologues of R14 and R15 in PTEN) are mutated to alanines in Ci-VSP, phosphatase activity is disrupted, as revealed by a lack of effect on the ionic currents of KCNQ2/3, where current decrease is a measure of phosphatase activity. The enzymatic activity was not rescued by the introduction of lysines, indicating that the binding is an arginine-specific interaction between the phosphatase binding domain and the membrane, presumably through the phosphate groups of the phospholipids. We also found that the kinetics and steady-state voltage dependence of the S4 segment movement are affected when the arginines are not present, indicating that the interaction of R253 and R254 with the membrane, required for the catalytic action of the phosphatase, restricts the movement of the voltage sensor.

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.

Postnatal maturation of the rat heart is characterized by major changes in the mechanism of excitation-contraction (E-C) coupling. In the neonate, the t tubules and sarcoplasmic reticulum (SR) are not fully developed yet. Consequently, Ca(2+)-induced Ca(2+) release (CICR) does not play a central role in E-C coupling. In the neonate, most of the Ca(2+) that triggers contraction comes through the sarcolemma. In this work, we defined the contribution of the sarcolemmal Ca(2+) entry and the Ca(2+) released from the SR to the Ca(2+) transient during the first 3 wk of postnatal development. To this end, intracellular Ca(2+) transients were measured in whole hearts from neonate rats by using the pulsed local field fluorescence technique. To estimate the contribution of each Ca(2+) flux to the global intracellular Ca(2+) transient, different pharmacological agents were used. Ryanodine was applied to evaluate ryanodine receptor-mediated Ca(2+) release from the SR, nifedipine for dihydropyridine-sensitive L-type Ca(2+) current, Ni(2+) for the current resulting from the reverse-mode Na(+)/Ca(2+) exchange, and mibefradil for the T-type Ca(2+) current. Our results showed that the relative contribution of each Ca(2+) flux changes considerably during the first 3 wk of postnatal development. Early after birth (1-5 days), the sarcolemmal Ca(2+) flux predominates, whereas at 3 wk of age, CICR from the SR is the most important. This transition may reflect the progressive development of the t tube-SR units characteristic of mature myocytes. We have hence directly defined in the whole beating heart the developmental changes of E-C coupling previously evaluated in single (acutely isolated or cultured) cells and multicellular preparations.