Nonconducting Mutant Research Articles

The anti-tumor drug 2-hydroxyoleic acid regulates the oncogenic potassium channel Kv10.1

Background2-hydroxyoleic acid (2OHOA) is a synthetic fatty acid with antitumor properties that alters membrane composition and structure, which in turn influences the functioning of membrane proteins and cell signaling. In this study, we propose a novel antitumoral mechanism of 2OHOA accomplished through the regulation of Kv10.1 channels. We evaluated the effects of 2OHOA on Kv10.1 channels expressed in HEK-293 cells by using electrophysiological techniques and a cell proliferation assay.Results2OHOA increased Kv10.1 channel currents in a voltage-dependent manner, shifted its conductance-voltage relationship towards negative potentials, and accelerated its activation kinetics. Moreover, 2OHOA reduced proliferation of cells that exogenously (HEK-293) and endogenously (MCF-7) expressed Kv10.1 channels. It is worth noting that the antiproliferative effect of 2OHOA was maintained in HEK-293 cells expressing a non-conducting mutant of Kv10.1 channel (Kv10.1-F456A), while it did not affect HEK-293 cells not expressing Kv10.1 channels, suggesting that 2OHOA interferes with a non-conducting function of Kv10.1 channels involved in cell proliferation. Finally, we found that 2OHOA can act synergistically with astemizole, a Kv10.1 channel blocker, to decrease cell proliferation more efficiently.ConclusionOur data suggest that 2OHOA decreases cell proliferation, at least in part, by regulating Kv10.1 channels.

Cryo-EM structure of the mammalian Kv2.1 channel and epileptic encephalopathy mutations provide insight into electromechanical coupling and slow inactivation.

The Kv2.1 delayed-rectifier K+ channel is one of the most abundant ion channels in the mammalian brain, where it is critical for neuronal activity, with mutations in humans causing epileptic encephalopathies. Kv2.1 opens slowly in response to membrane depolarization and slowly inactivates on the timescale of seconds. Slow inactivation in Kv2.1 is not sensitive to several manipulations that strongly influence C-type inactivation in Kv1 channels, including raising the concentration of external K+, external blockers such as tetraethylammonium (TEA) and mutations in the external pore near the ion selectivity filter. We solved the cryo-EM structure of Kv2.1 at 2.9-Å resolution, which reveals that the protein adopts an overall fold that is similar to Kv1 channels. Mutations causing epileptic encephalopathies are distributed throughout the structure of the transmembrane region of Kv2.1, producing distinct alterations in voltage-dependent activation. One particularly interesting epileptic encephalopathy mutant of a Phe to Leu near the internal end of the S6 pore-lining helix, rendered the channel effectively non-conducting; gating currents arising from activation of the voltage sensors could be measured without any evidence of ion conduction. From the cryo-EM structure, we identified a cluster of three Leu residues that interact intimately with the Phe, and when mutated to Ala remained conducting but with greatly accelerated (10-100-fold) slow inactivation. Experiments using internal TEA to detect opening of the internal gate in the non-conducting mutant of Kv2.1 suggest that the internal gate remains closed. From these and other findings, we propose that slow inactivation in Kv2.1 involves electromechanical coupling between the voltage sensors and pore, with reclosure of the internal gate occurring while the voltage sensors remain activated.

Role of C-Terminal Domain and Membrane Potential in the Mobility of Kv1.3 Channels in Immune Synapse Forming T Cells.

Voltage-gated Kv1.3 potassium channels are essential for maintaining negative membrane potential during T-cell activation. They interact with membrane-associated guanylate kinases (MAGUK-s) via their C-terminus and with TCR/CD3, leading to enrichment at the immunological synapse (IS). Molecular interactions and mobility may impact each other and the function of these proteins. We aimed to identify molecular determinants of Kv1.3 mobility, applying fluorescence correlation spectroscopy on human Jurkat T-cells expressing WT, C-terminally truncated (ΔC), and non-conducting mutants of mGFP-Kv1.3. ΔC cannot interact with MAGUK-s and is not enriched at the IS, whereas cells expressing the non-conducting mutant are depolarized. Here, we found that in standalone cells, mobility of ΔC increased relative to the WT, likely due to abrogation of interactions, whereas mobility of the non-conducting mutant decreased, similar to our previous observations on other membrane proteins in depolarized cells. At the IS formed with Raji B-cells, mobility of WT and non-conducting channels, unlike ΔC, was lower than outside the IS. The Kv1.3 variants possessing an intact C-terminus had lower mobility in standalone cells than in IS-engaged cells. This may be related to the observed segregation of F-actin into a ring-like structure at the periphery of the IS, leaving much of the cell almost void of F-actin. Upon depolarizing treatment, mobility of WT and ΔC channels decreased both in standalone and IS-engaged cells, contrary to non-conducting channels, which themselves caused depolarization. Our results support that Kv1.3 is enriched at the IS via its C-terminal region regardless of conductivity, and that depolarization decreases channel mobility.

PH Dependence of a Monomeric Non-Conducting Voltage-Gated Proton Channel (HV1)

The voltage-gated proton channel (Hv1) is a transmembrane protein made of a dimer of four transmembrane segments. This structure is capable of sensing the voltage and conforms the permeation pathway to protons. One of the most striking biophysical properties of Hv1 is its pH dependence of gating. The conductance-voltage (G-V) curve of Hv1 shifts depending on the pH gradient established across the membrane, but the molecular mechanism of this property is still poorly understood. Recently, gating currents of Hv1 were characterized using the low-conducting N264R mutant, giving us a new tool to understand the mechanism behind the pH dependence of Hv1. We measured currents produced by the monomeric N264R mutant of Ciona intestinalis Hv1 at different internal and external pH in membrane patches of Xenopus laevis oocytes. Although results showed a clear pH dependence of the gating currents, the presence of proton currents and charge trapping produced by the N264R mutation prevent a detailed analysis of the data. To eliminate proton currents, we measured gating currents produced by a monomeric non-conducting Hv1. Gating currents of the non-conducting mutant are free of proton current contamination and charge trapping. They preserve all the characteristics of Hv1 gating currents described previously. The charge-voltage (Q-V) curve is shifted to the left at lower ΔpH (pHin-pHout). Additionally, the ON-gating current decay is faster when ΔpH decreases. Our results show that the non-conducting mutant is a better choice to study pH dependence of Hv1 and understand the molecular mechanism behind this biophysical property. Supported by CONICYT-PFCHA/Doctorado Nacional/2017-21170395 to E.C., Fondecyt 1180464 to C.G. and 1150273 to R.L. The Centro Interdisciplinario de Neurociencia de Valparaiso is a Millennium Institute supported by the Millennium Scientific Initiative of the Chilean Ministry of Economy, Development, and Tourism (P029-022-F).

Molecular Mechanism of Depolarization-Dependent Inactivation in W366F Mutant of Kv1.2.

Voltage-gated potassium channels play crucial roles in regulating membrane potential. They are activated by membrane depolarization, allowing the selective permeation of K+ ions across the plasma membrane, and enter a nonconducting state after lasting depolarization, a process known as inactivation. Inactivation in voltage-activated potassium channels occurs through two distinct mechanisms, N-type and C-type inactivation. C-type inactivation is caused by conformational changes in the extracellular mouth of the channel, whereas N-type inactivation is elicited by changes in the cytoplasmic mouth of the protein. The W434F-mutated Shaker channel is known as a nonconducting mutant and is in a C-type inactivation state at a depolarizing membrane potential. To clarify the structural properties of C-type inactivated protein, we performed molecular dynamics simulations of the wild-type and W366F (corresponding to W434F in Shaker) mutant of the Kv1.2-2.1 chimera channel. The W366F mutant was in a nearly nonconducting state with a depolarizing voltage and recovered from inactivation with a reverse voltage. Our simulations and three-dimensional reference interaction site model analysis suggested that structural changes in the selectivity filter upon membrane depolarization trap K+ ions around the inner mouth of the selectivity filter and prevent ion permeation. This pore restriction is involved in the molecular mechanism of C-type inactivation.

On optimizing therapies, Orai, and ORNs

On optimizing therapies, Orai, and ORNs

Canonical Transient Receptor Potential Channel 2 (TRPC2) as a Major Regulator of Calcium Homeostasis in Rat Thyroid FRTL-5 Cells: IMPORTANCE OF PROTEIN KINASE C δ (PKCδ) AND STROMAL INTERACTION MOLECULE 2 (STIM2)

Mammalian non-selective transient receptor potential cation channels (TRPCs) are important in the regulation of cellular calcium homeostasis. In thyroid cells, including rat thyroid FRTL-5 cells, calcium regulates a multitude of processes. RT-PCR screening of FRTL-5 cells revealed the presence of TRPC2 channels only. Knockdown of TRPC2 using shRNA (shTRPC2) resulted in decreased ATP-evoked calcium peak amplitude and inward current. In calcium-free buffer, there was no difference in the ATP-evoked calcium peak amplitude between control cells and shTRPC2 cells. Store-operated calcium entry was indistinguishable between the two cell lines. Basal calcium entry was enhanced in shTRPC2 cells, whereas the level of PKCβ1 and PKCδ, the activity of sarco/endoplasmic reticulum Ca(2+)-ATPase, and the calcium content in the endoplasmic reticulum were decreased. Stromal interaction molecule (STIM) 2, but not STIM1, was arranged in puncta in resting shTRPC2 cells but not in control cells. Phosphorylation site Orai1 S27A/S30A mutant and non-functional Orai1 R91W attenuated basal calcium entry in shTRPC2 cells. Knockdown of PKCδ with siRNA increased STIM2 punctum formation and enhanced basal calcium entry but decreased sarco/endoplasmic reticulum Ca(2+)-ATPase activity in wild-type cells. Transfection of a truncated, non-conducting mutant of TRPC2 evoked similar results. Thus, TRPC2 functions as a major regulator of calcium homeostasis in rat thyroid cells.

Basis for allosteric open-state stabilization of voltage-gated potassium channels by intracellular cations

The open state of voltage-gated potassium (Kv) channels is associated with an increased stability relative to the pre-open closed states and is reflected by a slowing of OFF gating currents after channel opening. The basis for this stabilization is usually assigned to intrinsic structural features of the open pore. We have studied the gating currents of Kv1.2 channels and found that the stabilization of the open state is instead conferred largely by the presence of cations occupying the inner cavity of the channel. Large impermeant intracellular cations such as N-methyl-d-glucamine (NMG+) and tetraethylammonium cause severe slowing of channel closure and gating currents, whereas the smaller cation, Cs+, displays a more moderate effect on voltage sensor return. A nonconducting mutant also displays significant open state stabilization in the presence of intracellular K+, suggesting that K+ ions in the intracellular cavity also slow pore closure. A mutation in the S6 segment used previously to enlarge the inner cavity (Kv1.2-I402C) relieves the slowing of OFF gating currents in the presence of the large NMG+ ion, suggesting that the interaction site for stabilizing ions resides within the inner cavity and creates an energetic barrier to pore closure. The physiological significance of ionic occupation of the inner cavity is underscored by the threefold slowing of ionic current deactivation in the wild-type channel compared with Kv1.2-I402C. The data suggest that internal ions, including physiological concentrations of K+, allosterically regulate the deactivation kinetics of the Kv1.2 channel by impairing pore closure and limiting the return of voltage sensors. This may represent a primary mechanism by which Kv channel deactivation kinetics is linked to ion permeation and reveals a novel role for channel inner cavity residues to indirectly regulate voltage sensor dynamics.

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Initial steps in the opening of a Shaker potassium channel

The structural model of a K(V) (K(+)-selective, voltage-gated) channel in the open state is known (Protein Data Bank ID code 2R9R). Each subunit of the channel has four negatively charged residues distributed in the transmembrane segments S1, S2, and S3 that bind to and facilitate the movement within the membrane of the positively charged, voltage-sensing residues of S4. When extrapolated to the closed state, the two outermost negatively charged residues are exposed to extracellular fluid and not bound to S4 residues, all of which have theoretically been driven inward by voltage. If this closed state model is correct, these residues are available to bind external cations. We examined the effects of La(3+) on voltage-gated Shaker K(+) channels. Addition of the trivalent cation La(3+) (50 μM) extracellularly markedly prolongs the lag that precedes channel opening and slows the subsequent rise of K(+) current (I(K)) at all voltages. Decay kinetics of I(K) at negative voltages are unaltered. Gating current (I(g)) recorded from a nonconducting mutant shows that La(3+) reduces the initial amplitude of I(g) nearly twofold. We postulate that, in the resting state, La(3+) binds to the unoccupied, outermost negative residues, hindering outward S4 motion, thus increasing the lag on activation and slowing the rise of I(K). In the activated state, La(3+) is displaced by outward movement of arginine residues in S4; La(3+), therefore, is not present to affect channel closing. The results give strong support to the closed state model of the K(V) channel and a clear explanation of the effect of multivalent cations on cellular excitability.

Initial Opening Steps of K+ Channels Probed by Extracellular Multivalent Cations

The structural model of the open state of the Kv1.2-2.1 chimeric channel (Long et al., PDB 2R9R) has 4 negative residues distributed in S1, S2 and S3, all bound to positive residues in S4. Extrapolating the model to the resting state, the two outermost negative residues, E183 in S1 and E226 in S2, are exposed to the extracellular fluid and, in theory, not bound to S4 residues; E183 and E226 should thus be available to bind external cations. We have examined the effects on opening kinetics of multivalent cations using Shaker channels expressed in HEK cells. Addition of La3+ (50 μM) to the extracellular solution (in mM: 145 NaCl, 2 KCl, 10 CaCl2, pH 7.0) markedly prolonged the sigmoidal delay and slowed the rising phase of K+ current (IK) at all voltages tested. The closing kinetics of IK at negative voltages remained unaltered. Gating currents (Ig) recorded from a non-conducting mutant (W434F) showed that La3+ (50 μM) reduced the initial amplitude of Ig nearly twofold. We postulate that La3+ binds to the unoccupied negative side chains of E183 and E226, hindering outward S4 motion, thus increasing the lag and slowing the rise of IK. We further postulate that La3+, in the activated “up” state of S4, is not bound to E183 or E266, which are occupied by S4 positive charges; La3+ therefore has no effect on closing. Similar measurements using Ca2+ showed that at high concentrations it has an effect similar to that of La3+ on the activation time course. This Ca2+-dependent prolongation of the activation lag and slowing of the rising phase of IK is noticeably enhanced by complete removal of extracellular K+, perhaps because K+ and Ca2+ compete for E183 and E226 in the deactivated state.

Non-conducting function of the Kv2.1 channel enables it to recruit vesicles for release in neuroendocrine and nerve cells

Regulation of exocytosis by voltage-gated K(+) channels has classically been viewed as inhibition mediated by K(+) fluxes. We recently identified a new role for Kv2.1 in facilitating vesicle release from neuroendocrine cells, which is independent of K(+) flux. Here, we show that Kv2.1-induced facilitation of release is not restricted to neuroendocrine cells, but also occurs in the somatic-vesicle release from dorsal-root-ganglion neurons and is mediated by direct association of Kv2.1 with syntaxin. We further show in adrenal chromaffin cells that facilitation induced by both wild-type and non-conducting mutant Kv2.1 channels in response to long stimulation persists during successive stimulation, and can be attributed to an increased number of exocytotic events and not to changes in single-spike kinetics. Moreover, rigorous analysis of the pools of released vesicles reveals that Kv2.1 enhances the rate of vesicle recruitment during stimulation with high Ca(2+), without affecting the size of the readily releasable vesicle pool. These findings place a voltage-gated K(+) channel among the syntaxin-binding proteins that directly regulate pre-fusion steps in exocytosis.

Functional Interactions at the Interface between Voltage-Sensing and Pore Domains in the Shaker Kv Channel

Voltage-activated potassium (K(v)) channels contain a central pore domain that is partially surrounded by four voltage-sensing domains. Recent X-ray structures suggest that the two domains lack extensive protein-protein contacts within presumed transmembrane regions, but whether this is the case for functional channels embedded in lipid membranes remains to be tested. We investigated domain interactions in the Shaker K(v) channel by systematically mutating the pore domain and assessing tolerance by examining channel maturation, S4 gating charge movement, and channel opening. When mapped onto the X-ray structure of the K(v)1.2 channel the large number of permissive mutations support the notion of relatively independent domains, consistent with crystallographic studies. Inspection of the maps also identifies portions of the interface where residues are sensitive to mutation, an external cluster where mutations hinder voltage sensor activation, and an internal cluster where domain interactions between S4 and S5 helices from adjacent subunits appear crucial for the concerted opening transition.

Molecular determinants of the inhibition of human Kv1.5 potassium currents by external protons and Zn(2+).

Using human Kv1.5 channels expressed in HEK293 cells we assessed the ability of H+o to mimic the previously reported action of Zn(2+) to inhibit macroscopic hKv1.5 currents, and using site-directed mutagenesis, we addressed the mechanistic basis for the inhibitory effects of H(+)(o) and Zn(2+). As with Zn(2+), H(+)(o) caused a concentration-dependent, K(+)(o)-sensitive and reversible reduction of the maximum conductance (g(max)). With zero, 5 and 140 mM K(+)(o) the pK(H) for this decrease of g(max) was 6.8, 6.2 and 6.0, respectively. The concentration dependence of the block relief caused by increasing [K(+)](o) was well fitted by a non-competitive interaction between H(+)(o) and K(+)(o), for which the K(D) for the K(+) binding site was 0.5-1.0 mM. Additionally, gating current analysis in the non-conducting mutant hKv1.5 W472F showed that changing from pH 7.4 to pH 5.4 did not affect Q(max) and that charge immobilization, presumed to be due to C-type inactivation, was preserved at pH 5.4. Inhibition of hKv1.5 currents by H+o or Zn(2+) was substantially reduced by a mutation either in the channel turret (H463Q) or near the pore mouth (R487V). In light of the requirement for R487, the homologue of Shaker T449, as well as the block-relieving action of K(+)(o), we propose that H(+) or Zn(2+) binding to histidine residues in the pore turret stabilizes a channel conformation that is most likely an inactivated state.

Gating Charge Immobilization Caused by the Transition between Inactivated States in the Kv1.5 Channel

Sustained Na + or Li + conductance is a feature of the inactivated state in wild-type (WT) and nonconducting Shaker and Kv1.5 channels, and has been used here to investigate the cause of off-gating charge immobilization in WT and Kv1.5-W472F nonconducting mutant channels. Off-gating immobilization in response to brief pulses in cells perfused with NMG i +/NMG o + is the result of a more negative voltage dependence of charge recovery (V 1/2 is −96 mV) compared with on-gating charge movement (V 1/2 is −6.3 mV). This shift is known to be associated with slow inactivation in Shaker channels and the disparity is reduced by 40 mV, or ∼50% in the presence of 135 mM Cs i +. Off-gating charge immobilization is voltage-dependent with a V 1/2 of −12 mV, and correlates well with the development of Na + conductance on repolarization through C-type inactivated channels (V 1/2 is −11 mV). As well, the time-dependent development of the inward Na + tail current and gating charge immobilization after depolarizing pulses of different durations has the same time constant ( τ = 2.7 ms). These results indicate that in Kv1.5 channels the transition to a stable C-type inactivated state takes only 2–3 ms and results in strong charge immobilization in the absence of Group IA metal cations, or even in the presence of Na o +. Inclusion of low concentrations of Cs i + delays the appearance of Na + tail currents in WT channels, prevents transition to inactivated states in Kv1.5-W472F nonconducting mutant channels, and removes charge immobilization. Higher concentrations of Cs i + are able to modulate the deactivating transition in Kv1.5 channels and prevent the residual slowing of charge return.

Gating current studies reveal both intra- and extracellular cation modulation of K+ channel deactivation.

1. The presence of permeant ions can modulate the rate of gating charge return in wild-type human heart K+ (hKv1.5) channels. Here we employ gating current measurements in a non-conducting mutant, W472F, of the hKv1.5 channel to investigate how different cations can modulate charge return and whether the actions can be specifically localized at the internal as well as the external mouth of the channel pore. 2. Intracellular cations were effective at accelerating charge return in the sequence Cs+ > Rb+ > K+ > Na+ > NMG+. Extracellular cations accelerated charge return with the selectivity sequence Cs+ > Rb+ > Na+ = NMG+. 3. Intracellular and extracellular cation actions were of relatively low affinity. The Kd for preventing slowing of the time constant of the off-gating current decay (tau off) was 20.2 mM for intracellular Cs+ (Cs+i) and 358 mM for extracellular Cs+ (Cs+o). 4. Both intracellular and extracellular cations can regulate the rate of charge return during deactivation of hKv1.5, but intracellular cations are more effective. We suggest that ion crystal radius is an important determinant of this action, with larger ions preventing slowing more effectively. Important parallels exist with cation-dependent modulation of slow inactivation of ionic currents in this channel. However, further experiments are required to understand the exact relationship between acceleration of charge return and the slowing of inactivation of ionic currents by cations.

Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 alpha subunit.

A novel in vivo experimental strategy, involving cell type-specific expression of a dominant-negative K+ channel pore-forming alpha subunit, was developed and exploited to probe the molecular identity of the cardiac transient outward K+ current (I(to)). A point mutation (W to F) was introduced at position 362 in the pore region of Kv4.2 to produce a nonconducting mutant (Kv4.2W362F) subunit. Coexpression of Kv4.2W362F with Kv4.2 (or Kv4.3) attenuates the wild-type currents, and the effect is subfamily specific; ie, Kv4.2W362F does not affect heterologously expressed Kv1.4 currents. With the use of the alpha-myosin heavy chain promoter to direct cardiac-specific expression, several lines of Kv4.2W362F transgenic mice were generated. Electrophysiological recordings reveal that I(to) is selectively eliminated in ventricular myocytes isolated from transgenic mice expressing Kv4.2W362F, thereby demonstrating directly that the Kv 4 subfamily underlies I(to) in the mammalian heart. Functional knockout of I(to) leads to marked increases in action potential durations in ventricular myocytes and to prolongation of the QT interval in surface ECG recordings. In addition, a novel rapidly activating and inactivating K+ current, which is not detectable in myocytes from nontransgenic littermates, is evident in Kv4.2W362F-expressing ventricular cells. Importantly, these results demonstrate that electrical remodeling occurs in the heart when the expression of endogenous K- channels is altered.

Correlation between charge movement and ionic current during slow inactivation in Shaker K+ channels.

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.

Allosteric effects of permeating cations on gating currents during K+ channel deactivation.

K+ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K+ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K+ or Cs+, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K+ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs+ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at ECs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K+ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.

Molecular determinants of external barium block in Shaker potassium channels

Mutations in the outer pore region of Shaker K + channels ( T449 and D 447) can influence external Ba 2+ block. Substitution of T 449 by A, V or Y differentially reduced Ba 2+ block primarily by decreasing the blocking rate. Substitution of D 447 by N resulted in a non-conducting channel with apparently normal gating currents. External Ba 2+ can speed the OFF gating current of a different non-conducting mutant, W434F; this effect was markedly attenuated by the D447N substitution. These results suggest that D 447 contributes to an external Ba 2+ binding site while T 449 imposes a barrier to the access of that site.