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We previously proposed a role for the two-pore domain potassium (K2P) channel TREK-1 in hyperoxia (HO)-induced lung injury. To determine whether redundancy among the three TREK isoforms (TREK-1, TREK-2, and TRAAK) could protect from HO-induced injury, we now examined the effect of deletion of all three TREK isoforms in a clinically relevant scenario of prolonged HO exposure and mechanical ventilation (MV). We exposed WT and TREK-1/TREK-2/TRAAK-deficient [triple knockout (KO)] mice to either room air, 72-h HO, MV [high and low tidal volume (TV)], or a combination of HO + MV and measured quasistatic lung compliance, bronchoalveolar lavage (BAL) protein concentration, histologic lung injury scores (LIS), cellular apoptosis, and cytokine levels. We determined surfactant gene and protein expression and attempted to prevent HO-induced lung injury by prophylactically administering an exogenous surfactant (Curosurf). HO treatment increased lung injury in triple KO but not WT mice, including an elevated LIS, BAL protein concentration, and markers of apoptosis, decreased lung compliance, and a more proinflammatory cytokine phenotype. MV alone had no effect on lung injury markers. Exposure to HO + MV (low TV) further decreased lung compliance in triple KO but not WT mice, and HO + MV (high TV) was lethal for triple KO mice. In triple KO mice, the HO-induced lung injury was associated with decreased surfactant protein (SP) A and SPC but not SPB and SPD expression. However, these changes could not be explained by alterations in the transcription factors nuclear factor-1 (NF-1), NKX2.1/thyroid transcription factor-1 (TTF-1) or c-jun, or lamellar body levels. Prophylactic Curosurf administration did not improve lung injury scores or compliance in triple KO mice., (Copyright © 2017 the American Physiological Society.)

Ion channels are the cell's gatekeepers. These proteins selectively allow ionic current to flow down its electrochemical gradient. In some cases, specialized chemical or voltage sensing domains respond to environmental changes and signal the cell to adjust its internal chemistry in response to its surroundings. Because of their importance in cell function, channels have been the focus of intense study at the functional and structural level. Here we describe the optical technique voltage-clamp fluorometry (VCF) which is used to monitor the functional state and probe the structural rearrangements that take place as ion channels are activated by voltage. VCF combines electrophysiology, molecular biology, chemistry, and fluorescence into a single technique. Our focus is on voltage-gated ion channels, but the technique described can be applied to other proteins. We describe the cut open vaseline gap configuration (COVG) for VCF recording.

Neutralization of the aspartate near the selectivity filter in the GYGD pore sequence (D292N) of the voltage- and Ca(2+)-activated K+ channel (MaxiK, BKCa) does not prevent conduction like the corresponding mutation in Shaker channel, but profoundly affects major biophysical properties of the channel (Haug, T., D. Sigg, S. Ciani, L. Toro, E. Stefani, and R. Olcese. 2004. J. Gen. Physiol. 124:173-184). Upon depolarizations, the D292N mutant elicited mostly gating current, followed by small or no ionic current, at voltages where the wild-type hSlo channel displayed robust ionic current. In fact, while the voltage dependence of the gating current was not significantly affected by the mutation, the overall activation curve was shifted by approximately 20 mV toward more depolarized potentials. Several lines of evidence suggest that the mutation prevents population of certain open states that in the wild type lead to high open probability. The activation curves of WT and D292N can both be fitted to the sum of two Boltzmann distributions with identical slope factors and half activation potentials, just by changing their relative amplitudes. The steeper and more negative component of the activation curve was drastically reduced by the D292N mutation (from 0.65 to 0.30), suggesting that the population of open states that occurs early in the activation pathway is reduced. Furthermore, the slow component of the gating current, which has been suggested to reflect transitions from closed to open states, was greatly reduced in D292N channels. The D292N mutation also affected the limiting open probability: at 0 mV, the limiting open probability dropped from approximately 0.5 for the wild-type channel to 0.06 in D292N (in 1 mM [Ca2+]i). In addition to these effects on gating charge and open probability, as already described in Part I, the D292N mutation introduces a approximately 40% reduction of outward single channel conductance, as well as a strong outward rectification.

The pore region of the majority of K+ channels contains the highly conserved GYGD sequence, known as the K+ channel signature sequence, where the GYG is critical for K+ selectivity (Heginbotham, L., T. Abramson, and R. MacKinnon. 1992. Science. 258:1152-1155). Exchanging the aspartate residue with asparagine in this sequence abolishes ionic conductance of the Shaker K+ channel (D447N) (Hurst, R.S., L. Toro, and E. Stefani. 1996. FEBS Lett. 388:59-65). In contrast, we found that the corresponding mutation (D292N) in the pore forming alpha subunit (hSlo) of the voltage- and Ca(2+)-activated K+ channel (BKCa, MaxiK) did not prevent conduction but reduced single channel conductance. We have investigated the role of outer pore negative charges in ion conduction (this paper) and channel gating (Haug, T., R. Olcese, T. Ligia, and E. Stefani. 2004. J. Gen Physiol. 124:185-197). In symmetrical 120 mM [K+], the D292N mutation reduced the outward single channel conductance by approximately 40% and nearly abolished inward K+ flow (outward rectification). This rectification was partially relieved by increasing the external K+ concentration to 700 mM. Small inward currents were resolved by introducing an additional mutation (R207Q) that greatly increases the open probability of the channel. A four-state multi-ion pore model that incorporates the effects of surface charge was used to simulate the essential properties of channel conduction. The conduction properties of the mutant channel (D292N) could be predicted by a simple approximately 8.5-fold reduction of the surface charge density without altering any other parameter. These results indicate that the aspartate residue in the BKCa pore plays a key role in conduction and suggest that the pore structure is not affected by the mutation. We speculate that the negative charge strongly accumulates K+ in the outer vestibule close to the selectivity filter, thus increasing the rate of ion entry into the pore.

Animal and plant voltage-gated ion channels share a common architecture. They are made up of four subunits and the positive charges on helical S4 segments of the protein in animal K+ channels are the main voltage-sensing elements. The KAT1 channel cloned from Arabidopsis thaliana, despite its structural similarity to animal outward rectifier K+ channels is, however, an inward rectifier. Here we detected KAT1-gating currents due to the existence of an intrinsic voltage sensor in this channel. The measured gating currents evoked in response to hyperpolarizing voltage steps consist of a very fast (tau = 318 +/- 34 micros at -180 mV) and a slower component (4.5 +/- 0.5 ms at -180 mV) representing charge moved when most channels are closed. The observed gating currents precede in time the ionic currents and they are measurable at voltages (less than or equal to -60) at which the channel open probability is negligible ( approximately 10-4). These two observations, together with the fact that there is a delay in the onset of the ionic currents, indicate that gating charge transits between several closed states before the KAT1 channel opens. To gain insight into the molecular mechanisms that give rise to the gating currents and lead to channel opening, we probed external accessibility of S4 domain residues to methanethiosulfonate-ethyltrimethylammonium (MTSET) in both closed and open cysteine-substituted KAT1 channels. The results demonstrate that the putative voltage-sensing charges of S4 move inward when the KAT1 channels open.

Kv4 channels are thought to lack a C-type inactivation mechanism (collapse of the external pore) and to inactivate as a result of a concerted action of cytoplasmic regions of the channel. To investigate whether Kv4 channels have outer pore conformational changes during the inactivation process, the inactivation properties of Kv4.3 were characterized in 0 mM and in 2 mM external K+ in whole-cell voltage-clamp experiments. Removal of external K+ increased the inactivation rates and favored cumulative inactivation by repetitive stimulation. The reduction in current amplitude during repetitive stimulation and the faster inactivation rates in 0 mM external K+ were not due to changes in the voltage dependence of channel opening or to internal K+ depletion. The extent of the collapse of the K+ conductance upon removal of external K+ was more pronounced in NMG+-than in Na+-containing solutions. The reduction in the current amplitude during cumulative inactivation by repetitive stimulation is not associated with kinetic changes, suggesting that it is due to a diminished number of functional channels with unchanged gating properties. These observations meet the criteria for a typical C-type inactivation, as removal of external K+ destabilizes the conducting state, leading to the collapse of the pore. A tentative model is presented, in which K+ bound to high-affinity K+-binding sites in the selectivity filter destabilizes an outer neighboring K+ modulatory site that is saturated at approximately 2 mM external K+. We conclude that Kv4 channels have a C-type inactivation mechanism and that previously reported alterations in the inactivation rates after N- and C- termini mutagenesis may arise from secondary changes in the electrostatic interactions between K+-binding sites in the selectivity filter and the neighboring K+-modulatory site, that would result in changes in its K+ occupancy.

Kv4.3 channels are important molecular components of transient K(+) currents (Ito currents) in brain and heart. They are involved in setting the frequency of neuronal firing and heart pacing. Altered Kv4.3 channel expression has been demonstrated under pathological conditions like heart failure indicating their critical role in heart function. Thyroid hormone studies suggest that their expression in the heart may be hormonally regulated. To explore the possibility that sex hormones control Kv4.3 expression, we investigated whether its expression changes in the pregnant uterus. This organ represents a unique model to study Ito currents, because it possesses this type of K(+) current and undergoes dramatic changes in function and excitability during pregnancy. We cloned Kv4.3 channel from myometrium and found that its protein and transcript expression is greatly diminished during pregnancy. Experiments in ovariectomized rats demonstrate that estrogen is one mechanism responsible for the dramatic reduction in Kv4.3 expression and function prior to parturition. Furthermore, the reduction of plasma membrane Kv4.3 protein is accompanied by a perinuclear localization suggesting that cell trafficking is also controlled by sex hormones. Thus, estrogen remodels the expression of Kv4.3 in myometrium by directly diminishing its transcription and, indirectly, by altering Kv4.3 delivery to the plasma membrane.

In Shaker K(+) channel, the amino terminus deletion Delta6-46 removes fast inactivation (N-type) unmasking a slow inactivation process. In Shaker Delta6-46 (Sh-IR) background, two additional mutations (T449V-I470C) remove slow inactivation, producing a noninactivating channel. However, despite the fact that Sh-IR-T449V-I470C mutant channels remain conductive, prolonged depolarizations (1 min, 0 mV) produce a shift of the QV curve by about -30 mV, suggesting that the channels still undergo the conformational changes typical of slow inactivation. For depolarizations longer than 50 ms, the tail currents measured during repolarization to -90 mV display a slow component that increases in amplitude as the duration of the depolarizing pulse increases. We found that the slow development of the QV shift had a counterpart in the amplitude of the slow component of the ionic tail current that is not present in Sh-IR. During long depolarizations, the time course of both the increase in the slow component of the tail current and the change in voltage dependence of the charge movement could be well fitted by exponential functions with identical time constant of 459 ms. Single channel recordings revealed that after prolonged depolarizations, the channels remain conductive for long periods after membrane repolarization. Nonstationary autocovariance analysis performed on macroscopic current in the T449V-I470C mutant confirmed that a novel open state appears with increasing prepulse depolarization time. These observations suggest that in the mutant studied, a new open state becomes progressively populated during long depolarizations (>50 ms). An appealing interpretation of these results is that the new open state of the mutant channel corresponds to a slow inactivated state of Sh-IR that became conductive.

Large conductance voltage-dependent and Ca(2+)-modulated K(+) channels play a crucial role in myometrium contractility. Western blots and immunocytochemistry of rat uterine sections or isolated cells show that MaxiK channel protein signals drastically decrease towards the end of pregnancy. Consistent with a transcriptional regulation of channel expression, mRNA levels quantified with the ribonuclease protection assay correlated well with MaxiK protein levels. As a control, Na(+)/K(+)-ATPase protein and RNA levels do not significantly change at different stages of pregnancy. The low numbers of MaxiK channels at the end of pregnancy may facilitate uterine contraction needed for parturition.

Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as "charge immobilization" (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567-590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at -90 mV return potential changed from a single fast component to at least two components, the slower requiring approximately 200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current (Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at -120 and -90 mV. In contrast, at higher potentials (-70 and -50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of "parallel" inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH2-terminus region.

Prolonged depolarization induces a slow inactivation process in some K+ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Delta(6-46) K+ channel and in the nonconducting mutant (Shaker H4-Delta(6-46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547-556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to -90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non-slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K+ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011-1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.

Large conductance calcium- and voltage-sensitive K+ (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca2+ favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca2+ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4-5 elementary charges.

The energy profile of the interaction between the NH2-terminal inactivation domain and the internal mouth of the Shaker H4 K+ channel has been investigated. Macroscopic currents from channels normally inactivating (Shaker H4) and with the inactivation removed (Shaker H4-IR) were recorded at different temperatures using the cut-open oocyte technique. Changes in temperature had a dramatic effect on the inactivation phase. The following parameters were obtained in Shaker H4, lowering the temperature from 20 degrees C to 5 degrees C: (1) the peak amplitude decreased with the temperature coefficient Q10 equal to 1.51; (2) the activation time constant increased with a Q10 equal to 3.14; (3) the decay time constant increased with a Q10 of 7.20, while the recovery from inactivation was less temperature-dependent (Q10=1.57) than the installation of the inactivation phase. At 0 mV, the ratio between the steady state level and the peak amplitude of the current increased with a Q10 of 2.95. These findings indicate that the installation of a fast inactivation process has a strong temperature dependence, while the recovery phase from inactivation is less temperature dependent. These observations support the idea of an NH2-terminal blocking mechanism for inactivation and flexible conformation of the blocking particle.

This review summarizes recent advances in our understanding of how the BK channel transduces Ca2+and voltage signals into opening of the ion conduction gate to regulate K+flux. [ABSTRACT FROM AUTHOR]