Determinants of frequency-dependent regulation of Kv1.2-containing potassium channels

Voltage-gated potassium channels are important regulators of electrical excitation in many tissues, with Kv1.2 standing out as an essential contributor in the CNS. Genetic deletion of Kv1.2 invariably leads to early lethality in mice. In humans, mutations affecting Kv1.2 function are linked to epileptic encephalopathy and movement disorders. We have demonstrated that Kv1.2 is subject to a unique regulatory mechanism in which repetitive stimulation leads to dramatic potentiation of current. In this study, we explore the properties and molecular determinants of this use-dependent potentiation/activation. First, we examine how alterations in duty cycle (depolarization and repolarization/recovery times) affect the onset and extent of use-dependent activation. Also, we use trains of repetitive depolarizations to test the effects of a variety of Thr252 (S2-S3 linker) mutations on use-dependent activation. Substitutions of Thr with some sterically similar amino acids (Ser, Val, and Met, but not Cys) retain use-dependent activation, while bulky or charged amino acid substitutions eliminate use-dependence. Introduction of Thr at the equivalent position in other Kv1 channels (1.1, 1.3, 1.4), was not sufficient to transfer the phenotype. We hypothesize that use-dependent activation of Kv1.2 channels is mediated by an extrinsic regulator that binds preferentially to the channel closed state, with Thr252 being necessary but not sufficient for this interaction to alter channel function. These findings extend the conclusions of our recent demonstration of use-dependent activation of Kv1.2-containing channels in hippocampal neurons, by adding new details about the molecular mechanism underlying this effect.     

The pore region of the Kv1.2alpha subunit is an important component of recombinant Kv1.2 channel oxygen sensitivity

Oxygen-sensitive K(+) channels are important elements in the cellular response to hypoxia. Although much progress has been made in identifying their molecular composition, the structural components associated to their O(2)-sensitivity are not yet understood. Recombinant Kv1.2 currents expressed in Xenopus oocytes are inhibited by a decrease in O(2) availability. On the contrary, heterologous Kv2.1 channels are O(2)-insensitive. To elucidate the protein segment responsible for the O(2)-sensitivity of Kv1.2 channels, we analyzed the response to anoxia of Kv1.2/Kv2.1 chimeric channels. Expression of chimeric Kv2.1 channels each containing the S4, the S1-S3 or the S6-COOH segments of Kv1.2 polypeptide resulted in a K(+) current insensitive to anoxia. In contrast, transferring the S5-S6 segment of Kv1.2 into Kv2.1 produced an O(2)-sensitive K(+) current. Finally, mutating a redox-sensitive methionine residue (M380) of Kv1.2 polypeptide did not affect O(2)-sensitivity. Thus, the pore and its surrounding regions of Kv1.2 polypeptide confer its hypoxic inhibition. This response is independent on the redox modulation of methionine residues in this protein segment.     

Contribution of electrostatic and structural properties of Kv4.3 S4 arginine residues to the regulation of channel gating

Previous work has demonstrated that replacing individual arginine (R) residues in the S4 domain of Kv4.3 with alanine (A) not only altered activation and deactivation processes, but also those of closed-state inactivation (CSI) and recovery. R → A mutants eliminated individual positive charge while substantially reducing side chain volume and hydrophilic character. Their novel effects on gating may thus have been the result of electrostatic and/or structural perturbations. To address this issue, and to gain further insights into the roles that S4 plays in the regulation of Kv4.3 gating transitions, we comparatively analyzed arginine to glutamine (R → Q) mutations at positions 290, 293, and 296. This maneuver maintained positive charge elimination of the R → A mutants, while partially restoring native side chain volume and hydrophilic properties. R → A and R → Q mutant pairs produced similar effects on the forward gating process of activation. In contrast, significant differences between the two substitutions were discovered on deactivation, CSI, and recovery, with the R → Q mutants partially restoring wild type characteristics. Our results argue that modification of individual S4 residue properties may result in altered localized interactions within unique microenvironments encountered during forward and reverse gating transitions. As such, predominant effects appear on the reverse gating transitions of deactivation and recovery. These results are consistent with the proposal that arginine residues in S4 are involved in regulating Kv4.3 CSI and recovery.     

Electrostatic interaction in the NH2-terminus accelerates inactivation of the Kv1.4 channel

Inactivation of potassium channels plays an important role in shaping the electrical signalling properties of nerve and muscle cells. While it has been assumed that the rapid inactivation of the Kv1.4 channel is controlled by a “ball and chain” inactivation mechanism, the chain structure of the channel has not been well defined. Here, by conducting electrophysiological studies on variants containing mutations of the positively charged and negatively charged segments of the NH₂-terminal of the channel protein, we show that neutralization or deletion of the positively charged segment (residues 83-98) significantly slowed the inactivation process. Replacement of this positively charged segment with the negatively charged segment (residues 123-137), and vice versa, so that both segments were simultaneously positively or negatively charged, also slowed the inactivation process. Furthermore, the inactivation process was not changed when the positively charged and the negatively charged segments were interchanged. In contrast, the voltage dependence of activation and inactivation of the channels was not significantly altered by these mutants. These results indicate that the electrostatic interaction between the positively and negatively charged segments plays a critical role in the inactivation process of the Kv1.4 channel. Taken together, we propose that the electrostatic interaction accelerates the inactivation of the Kv1.4 channel by making it easier for the inactivation ball to access its binding site.     

Src regulates membrane trafficking of the Kv3.1b channel

The Kv3.1 channel plays a crucial role in regulating the high-frequency firing properties of neurons. Here, we determined whether Src regulates the subcellular distributions of the Kv3.1b channel. Co-expression of active Src induced a dramatic redistribution of Kv3.1b to the endoplasmic reticulum. Furthermore, co-expression of the Kv3.1b channel with active Src induced a remarkable decrease in the pool of Kv3.1b at the cell surface. Moreover, the co-expression of active Src results in a significant decrease in the peak current densities of the Kv3.1b channel, and a substantial alteration in the voltage dependence of its steady-state inactivation. Taken together, these results indicate that Src kinase may play an important role in regulating membrane trafficking of Kv3.1b channels.     

Membrane tension accelerates rate-limiting voltage-dependent activation and slow inactivation steps in a Shaker channel

A classical voltage-sensitive channel is tension sensitive—the kinetics of Shaker and S3–S4 linker deletion mutants change with membrane stretch (Tabarean, I.V., and C.E. Morris. 2002. Biophys. J. 82:2982–2994.). Does stretch distort the channel protein, producing novel channel states, or, more interestingly, are existing transitions inherently tension sensitive? We examined stretch and voltage dependence of mutant 5aa, whose ultra-simple activation (Gonzalez, C., E. Rosenman, F. Bezanilla, O. Alvarez, and R. Latorre. 2000. J. Gen. Physiol. 115:193–208.) and temporally matched activation and slow inactivation were ideal for these studies. We focused on macroscopic patch current parameters related to elementary channel transitions: maximum slope and delay of current rise, and time constant of current decline. Stretch altered the magnitude of these parameters, but not, or minimally, their voltage dependence. Maximum slope and delay versus voltage with and without stretch as well as current rising phases were well described by expressions derived for an irreversible four-step activation model, indicating there is no separate stretch-activated opening pathway. This model, with slow inactivation added, explains most of our data. From this we infer that the voltage-dependent activation path is inherently stretch sensitive. Simulated currents for schemes with additional activation steps were compared against datasets; this showed that generally, additional complexity was not called for. Because the voltage sensitivities of activation and inactivation differ, it was not possible to substitute depolarization for stretch so as to produce the same overall P_O time course. What we found, however, was that at a given voltage, stretch-accelerated current rise and decline almost identically—normalized current traces with and without stretch could be matched by a rescaling of time. Rate-limitation of the current falling phase by activation was ruled out. We hypothesize, therefore, that stretch-induced bilayer decompression facilitates an in-plane expansion of the protein in both activation and inactivation. Dynamic structural models of this class of channels will need to take into account the inherent mechanosensitivity of voltage-dependent gating.     

A specific N-terminal residue in Kv1.5 is required for upregulation of the channel by SAP97

We have previously reported that SAP97 enhancement of hKv1.5 currents requires an intact Kv1.5 N-terminus and is independent of the PDZ-binding motif at the C-terminus of the channel [J. Eldstrom, W.S. Choi, D.F. Steele, D. Fedida, SAP97 increases Kv1.5 currents through an indirect N-terminal mechanism, FEBS Lett. 547 (2003) 205-211]. Here, we report that an interaction between the two proteins can be detected under certain conditions but their interaction is irrelevant to the enhancement of channel expression. Instead, a threonine residue at position 15 in the hKv1.5 N-terminus is critically important. Mutation of this residue, which lies within a consensus site for phosphorylation by protein kinase C, to an alanine, completely abrogated the effect of SAP97 on channel expression. Although we were unable to detect phosphorylation of this residue, specific inhibition of kinase C by Calphostin C eliminated the increase in wild-type hKv1.5 currents associated with SAP97 overexpression suggesting a role for this kinase in the response.     

Constitutive activation of the Shaker Kv channel

In different types of K+ channels the primary activation gate is thought to reside near the intracellular entrance to the ion conduction pore. In the Shaker Kv channel the gate is closed at negative membrane voltages, but can be opened with membrane depolarization. In a previous study of the S6 activation gate in Shaker (Hackos, D.H., T.H. Chang, and K.J. Swartz. 2002. J. Gen. Physiol. 119:521-532.), we found that mutation of Pro 475 to Asp results in a channel that displays a large macroscopic conductance at negative membrane voltages, with only small increases in conductance with membrane depolarization. In the present study we explore the mechanism underlying this constitutively conducting phenotype using both macroscopic and single-channel recordings, and probes that interact with the voltage sensors or the intracellular entrance to the ion conduction pore. Our results suggest that constitutive conduction results from a dramatic perturbation of the closed-open equilibrium, enabling opening of the activation gate without voltage-sensor activation. This mechanism is discussed in the context of allosteric models for activation of Kv channels and what is known about the structure of this critical region in K+ channels.     

AMIGO is an auxiliary subunit of the Kv2.1 potassium channel

Kv2.1 is a potassium channel α-subunit abundantly expressed throughout the brain. It is a main component of delayed rectifier current (I(K)) in several neuronal types and a regulator of excitability during high-frequency firing. Here we identify AMIGO (amphoterin-induced gene and ORF), a neuronal adhesion protein with leucine-rich repeat and immunoglobin domains, as an integral part of the Kv2.1 channel complex. AMIGO shows extensive spatial and temporal colocalization and association with Kv2.1 in the mouse brain. The colocalization of AMIGO and Kv2.1 is retained even during stimulus-induced changes in Kv2.1 localization. AMIGO increases Kv2.1 conductance in a voltage-dependent manner in HEK cells. Accordingly, inhibition of endogenous AMIGO suppresses neuronal I(K) at negative membrane voltages. In conclusion, our data indicate AMIGO as a function-modulating auxiliary subunit for Kv2.1 and thus provide new insights into regulation of neuronal excitability.     

Proteomic analysis highlights the molecular complexities of native Kv4 channel macromolecular complexes

Voltage-gated K(+) (Kv) channels are key determinants of membrane excitability in the nervous and cardiovascular systems, functioning to control resting membrane potentials, shape action potential waveforms and influence the responses to neurotransmitters and neurohormones. Consistent with this functional diversity, multiple types of Kv currents, with distinct biophysical properties and cellular/subcellular distributions, have been identified. Rapidly activating and inactivating Kv currents, typically referred to as I(A) (A-type) in neurons, for example, regulate repetitive firing rates, action potential back-propagation (into dendrites) and modulate synaptic responses. Currents with similar properties, referred to as I(to,f) (fast transient outward), expressed in cardiomyocytes, control the early phase of myocardial action potential repolarization. A number of studies have demonstrated critical roles for pore-forming (α) subunits of the Kv4 subfamily in the generation of native neuronal I(A) and cardiac I(to,f) channels. Studies in heterologous cells have also suggested important roles for a number of Kv channel accessory and regulatory proteins in the generation of functional I(A) and I(to,f) channels. Quantitative mass spectrometry-based proteomic analysis is increasingly recognized as a rapid and, importantly, unbiased, approach to identify the components of native macromolecular protein complexes. The recent application of proteomic approaches to identify the components of native neuronal (and cardiac) Kv4 channel complexes has revealed even greater complexity than anticipated. The continued emphasis on development of improved biochemical and analytical proteomic methods seems certain to accelerate progress and to provide important new insights into the molecular determinants of native ion channel protein complexes.     

Structural determinants for the action of grayanotoxin in D1 S4-S5 and D4 S4-S5 intracellular linkers of sodium channel alpha-subunits

We located a novel binding site for grayanotoxin on the cytoplasmic linkers of voltage-dependent cardiac (rH1) or skeletal-muscle (mu 1) Na(+) channel isoforms (segments S4-S5 in domains D1 and D4), using the alanine scanning substitution method. GTX-modification of Na(+) channels, transiently expressed in HEK 293 cells, was evaluated under whole-cell voltage clamp, from the ratio of maximum chord conductance for modified and unmodified Na(+) channels. In mu 1, mutations K237A, L243A, S246A, K248A, K249A, L250A, S251A, or T1463A, caused a moderate, but statistically significant decrease in this ratio. On making corresponding mutations in rH1, only L244A dramatically reduced the ratio. Because in mu 1, the serine at position 251 is the only heterologous residue with respect to rH1 (Ala-252), we made a double mutant L243A&S251A to match the sequence of mu 1 and rH1 in S4-S5 linkers of both domains. This double mutation resulted in a significant decrease in the ratio, to the same extent as L244A substitution in rH1 did, indicating that the site at Leu-244 in rH1 or at Leu-243 in mu 1 is a novel one, exhibiting a synergistic effect of grayanotoxin.     

Modulation of the Shaker K(+) channel gating kinetics by the S3-S4 linker

In Shaker K(+) channels depolarization displaces outwardly the positively charged residues of the S4 segment. The amount of this displacement is unknown, but large movements of the S4 segment should be constrained by the length and flexibility of the S3-S4 linker. To investigate the role of the S3-S4 linker in the ShakerH4Delta(6-46) (ShakerDelta) K(+) channel activation, we constructed S3-S4 linker deletion mutants. Using macropatches of Xenopus oocytes, we tested three constructs: a deletion mutant with no linker (0 aa linker), a mutant containing a linker 5 amino acids in length, and a 10 amino acid linker mutant. Each of the three mutants tested yielded robust K(+) currents. The half-activation voltage was shifted to the right along the voltage axis, and the shift was +45 mV in the case of the 0 aa linker channel. In the 0 aa linker, mutant deactivation kinetics were sixfold slower than in ShakerDelta. The apparent number of gating charges was 12.6+/-0.6 e(o) in ShakerDelta, 12.7+/-0.5 in 10 aa linker, and 12.3+/-0.9 in 5 aa linker channels, but it was only 5.6+/-0.3 e(o) in the 0 aa linker mutant channel. The maximum probability of opening (P(o)(max)) as measured using noise analysis was not altered by the linker deletions. Activation kinetics were most affected by linker deletions; at 0 mV, the 5 and 0 aa linker channels' activation time constants were 89x and 45x slower than that of the ShakerDelta K(+) channel, respectively. The initial lag of ionic currents when the prepulse was varied from -130 to -60 mV was 0.5, 14, and 2 ms for the 10, 5, and 0 aa linker mutant channels, respectively. These results suggest that: (a) the S4 segment moves only a short distance during activation since an S3-S4 linker consisting of only 5 amino acid residues allows for the total charge displacement to occur, and (b) the length of the S3-S4 linker plays an important role in setting ShakerDelta channel activation and deactivation kinetics.     

Role of the S2 and S3 Segment in Determining the Activation Kinetics in Kv2.1 Channels

We constructed chimeras between the rapidly activating Kv1.2 channel and the slowly activating Kv2.1 channel in order to study to what extent sequence differences within the S1–S4 region contribute to the difference in activation kinetics. The channels were expressed in Xenopus oocytes and the currents were measured with a two-microelectrode voltage-clamp technique. Substitution of the S1–S4 region of Kv2.1 subunits by the ones of Kv1.2 resulted in chimeric channels which activated more rapidly than Kv2.1. Furthermore, activation kinetics were nearly voltage-independent in contrast to the pronounced voltage-dependent activation kinetics of both parent channels. Systematic screening of the S1–S4 region by the replacement of smaller protein parts resolved that the main functional changes generated by the S1–S4 substitution were generated by the S2 and the S3 segment. However, the effects of these segments were different: The S3 substitution reduced the effective gating charge and accelerated both a voltage-dependent and a voltage-independent component of the activation time course. In contrast, the S2 substitution accelerated predominantly the voltage-dependent component of the activation time course thereby leaving the effective gating charge unchanged. It is concluded that the S2 and the S3 segment determine the activation kinetics in a specific manner. Received: 13 November 2000/Revised: 5 April 2001     

Ceramide inhibits the potassium channel Kv1.3 by the formation of membrane platforms

Previous studies suggested a central role of sphingomyelin- and cholesterol-enriched membrane rafts in the initiation of signaling via many receptors. Here, we investigated the role of membrane rafts for the function of the voltage-gated potassium channel Kv1.3. We demonstrate that Kv1.3 localizes in the cell membrane to pre-existing small, sphingolipid- and cholesterol-enriched membrane rafts. Transformation of these small rafts to large ceramide-enriched membrane platforms was achieved by stimulation of the endogenous acid sphingomyelinase, addition of exogenous sphingomyelinase or treatment of the cells with C(16)-ceramide and resulted in clustering of Kv1.3 within ceramide-enriched membrane platforms and inhibition of the channel's activity. Likewise, disruption of pre-existing small rafts inhibited Kv1.3 activity. This indicates that intact small membrane rafts are required for Kv1.3 activity and an alteration of the lipid environment of rafts inhibits Kv1.3. These data, thus, may suggest a novel concept for the regulation of ion channels by the cell membrane composition.     

Ca2+- and voltage-dependent inactivation of the expressed L-type Ca(v)1.2 calcium channel

Ca2+-dependent regulation of the ion current through the alpha1Cbeta2aalpha2delta-1 (L-type) calcium channel transiently expressed in HEK 293 cells was investigated using whole cell patch clamp method. Ca2+ or Na+ ions were used as a charge carrier. Intracellular Ca2+ was either buffered by 10 mM EGTA or 200 microM Ca2+ was added into non-buffered intracellular solution. Free intracellular Ca2+ inactivated permanently about 80% of the L-type calcium current. The L-type calcium channel inactivated during a depolarizing pulse with two time constants, tau(fast) and tau(slow). Free intracellular calcium accelerated both time constants. Effect on the tau(slow) was more pronounced. About 80% of the channel inactivation during brief depolarizing pulse could be attributed to a Ca2+-dependent mechanism and 20% to a voltage-dependent mechanism. When Na+ ions were used as a charge carrier, the L-type current still inactivated with two time constants that were 10 times slower and were virtually voltage-independent. Ca2+ ions stabilized the inactivated state of the channel in a concentration-dependent manner.     

Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain

Chronic pain states are characterized by long-term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up-regulation of Cav1.2-comprising L-type calcium channel (Cav1.2-LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR-103, simultaneously regulates the expression of the three subunits forming Cav1.2-LTC in a novel integrative regulation. This regulation is bidirectional since knocking-down or over-expressing miR-103, respectively, up- or down-regulate the level of Cav1.2-LTC translation. Functionally, we show that miR-103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR-103 is down-regulated in neuropathic animals and that miR-103 intrathecal applications successfully relieve pain, identifying miR-103 as a novel possible therapeutic target in neuropathic chronic pain.     

Sensitivity of the Drosophila para sodium channel to DDT is not lowered by the super-kdr mutation M918T on the IIS4-S5 linker that profoundly reduces sensitivity to permethrin and deltamethrin

DDT inhibits Na channel inactivation and deactivation, promotes Na channel activation and reduces the resting potential of Xenopus oocytes expressing the Drosophila para Na channel. These changes are only marginally influenced by the single mutation M918T (super-kdr) but are reduced approximately 10-fold by either the single mutation L1014F (kdr) or the double mutation L1014F+M918T, both of which confer resistance to the pyrethroids permethrin and deltamethrin. We conclude that DDT binds either to or in the region of L1014 on IIS6 but only weakly to M918 on the IIS4-S5 linker, which is part of a high-affinity binding site for permethrin and deltamethrin.     

Uncoupling of gating charge movement and closure of the ion pore during recovery from inactivation in the Kv1.5 channel

Both wild-type (WT) and nonconducting W472F mutant (NCM) Kv1.5 channels are able to conduct Na(+) in their inactivated states when K(+) is absent. Replacement of K(+) with Na(+) or NMG(+) allows rapid and complete inactivation in both WT and W472F mutant channels upon depolarization, and on return to negative potentials, transition of inactivated channels to closed-inactivated states is the first step in the recovery of the channels from inactivation. The time constant for immobilized gating charge recovery at -100 mV was 11.1 +/- 0.4 ms (n = 10) and increased to 19.0 +/- 1.6 ms (n = 3) when NMG(+)(o) was replaced by Na(+)(o). However, the decay of the Na(+) tail currents through inactivated channels at -100 mV had a time constant of 129 +/- 26 ms (n = 18), much slower than the time required for gating charge recovery. Further experiments revealed that the voltage-dependence of gating charge recovery and of the decay of Na(+) tail currents did not match over a 60 mV range of repolarization potentials. A faster recovery of gating charge than pore closure was also observed in WT Kv1.5 channels. These results provide evidence that the recovery of the gating elements is uncoupled from that of the pore in Na(+)-conducting inactivated channels. The dissociation of the gating charge movements and the pore closure could also be observed in the presence of symmetrical Na(+) but not symmetrical Cs(+). This difference probably stems from the difference in the respective abilities of the two ions to limit inactivation to the P-type state or prevent it altogether.