Elucidating KChiP effects on Kv4.3 inactivation and recovery kinetics with a minimal KChiP2 isoform

Kv channel interacting proteins (KChIPs) are Ca²⁺-binding proteins with four EF-hands. KChIPs modulate Kv4 channel gating by slowing inactivation kinetics and accelerating recovery kinetics. Thus, KChIPs are believed to be important regulators of Kv4 channels underlying transient outward K⁺ currents in many excitable cell types. We have cloned a structurally minimal KChIP2 isoform (KChIP2d) from ferret heart. KChIP2d corresponds to the final 70 C-terminal amino acids of other KChIPs and has only one EF-hand. We demonstrate that KChIP2d is a functional KChIP that both accelerates recovery and slows inactivation kinetics of Kv4.3, indicating that the minimal C-terminus can maintain KChIP regulatory properties. We utilize KChIP2d to further demonstrate that: (i) the EF-hand modulates effects on Kv4.3 inactivation but not recovery; (ii) Ca²⁺-dependent effects on Kv4.3 inactivation are mediated through a mechanism reflected in the slow time constant of inactivation; and (iii) a short stretch of amino acids exclusive of the EF-hand partially mediates Ca²⁺-independent effects on recovery. Our results demonstrate that distinct regions of a KChIP molecule are involved in modulating inactivation and recovery. The potential ability of KChIP EF-hands to sense intracellular Ca²⁺ levels and transduce these changes to alterations in Kv4 channel inactivation kinetics may serve as a mechanism allowing intracellular Ca²⁺ transients to modulate repolarization. KChIP2d is a valuable tool for elucidating structural domains of KChIPs involved in Kv4 channel regulation.     

Activation properties of Kv4.3 channels: time, voltage and [K+]o dependence

Rapidly inactivating, voltage-dependent K⁺ currents play important roles in both neurones and cardiac myocytes. Kv4 channels form the basis of these currents in many neurones and cardiac myocytes and their mechanism of inactivation appears to differ significantly from that reported for Shaker and Kv1.4 channels. In most channel gating models, inactivation is coupled to the kinetics of activation. Hence, there is a need for a rigorous model based on comprehensive experimental data on Kv4.3 channel activation. To develop a gating model of Kv4.3 channel activation, we studied the properties of Kv4.3 channels in Xenopus oocytes, without endogenous KChIP2 ancillary subunits, using the perforated cut-open oocyte voltage clamp and two-electrode voltage clamp techniques. We obtained high-frequency resolution measurements of the activation and deactivation properties of Kv4.3 channels. Activation was sigmoid and well described by a fourth power exponential function. The voltage dependence of the activation time constants was best described by a biexponential function corresponding to at least two different equivalent charges for activation. Deactivation kinetics are voltage dependent and monoexponential. In contrast to other voltage-sensitive K⁺ channels such as HERG and Shaker , we found that elevated extracellular [K⁺] modulated the activation process by slowing deactivation and stabilizing the open state. Using these data we developed a model with five closed states and voltage-dependent transitions between the first four closed states coupled to a voltage-insensitive transition between the final closed (partially activated) state and the open state. Our model closely simulates steady-state and kinetic activation and deactivation data.     

Remodelling inactivation gating of Kv4 channels by KChIP1, a small-molecular-weight calcium-binding protein

Calcium-binding proteins dubbed KChIPs favour surface expression and modulate inactivation gating of neuronal and cardiac A-type Kv4 channels. To investigate their mechanism of action, Kv4.1 or Kv4.3 were expressed in Xenopus laevis oocytes, either alone or together with KChIP1, and the K⁺ currents were recorded using the whole-oocyte voltage-clamp and patch-clamp methods. KChIP1 similarly remodels gating of both channels. At positive voltages, KChIP1 slows the early phase of the development of macroscopic inactivation. By contrast, the late phase is accelerated, which allows complete inactivation in < 500 ms. Thus, superimposed traces from control and KChIP1-remodelled currents crossover. KChIP1 also accelerates closed-state inactivation and recovery from inactivation (3- to 5-fold change). The latter effect is dominating and, consequently, the prepulse inactivation curves exhibit depolarizing shifts (Δ V = 4–12 mV). More favourable closed-state inactivation may also contribute to the overall faster inactivation at positive voltages because Kv4 channels significantly inactivate from the preopen closed state. KChIP1 favours this pathway further by accelerating channel closing. The peak G-V curves are modestly leftward shifted in the presence of KChIP1, but the apparent 'threshold' voltage of current activation remains unaltered. Single Kv4.1 channels exhibited multiple conductance levels that ranged between 1.8 and 5.6 pS in the absence of KChIP1 and between 1.9 and 5.3 pS in its presence. Thus, changes in unitary conductance do not contribute to current upregulation by KChIP1. An allosteric kinetic model explains the kinetic changes by assuming that KChIP1 mainly impairs open-state inactivation, favours channel closing and lowers the energy barrier of closed-state inactivation.     

Ca2+-activated K+ (BK) channel inactivation contributes to spike broadening during repetitive firing in the rat lateral amygdala

In many neurons, trains of action potentials show frequency-dependent broadening. This broadening results from the voltage-dependent inactivation of K⁺ currents that contribute to action potential repolarisation. In different neuronal cell types these K⁺ currents have been shown to be either slowly inactivating delayed rectifier type currents or rapidly inactivating A-type voltage-gated K⁺ currents. Recent findings show that inactivation of a Ca²⁺-dependent K⁺ current, mediated by large conductance BK-type channels, also contributes to spike broadening. Here, using whole-cell recordings in acute slices, we examine spike broadening in lateral amygdala projection neurons. Spike broadening is frequency dependent and is reversed by brief hyperpolarisations. This broadening is reduced by blockade of voltage-gated Ca²⁺ channels and BK channels. In contrast, broadening is not blocked by high concentrations of 4-aminopyridine (4-AP) or α-dendrotoxin. We conclude that while inactivation of BK-type Ca²⁺-activated K⁺ channels contributes to spike broadening in lateral amygdala neurons, inactivation of another as yet unidentified outward current also plays a role.     

Separation of P/C- and U-type inactivation pathways in Kv1.5 potassium channels

P/C-type inactivation of Kv channels is thought to involve conformational changes in the outer pore of the channel, culminating in a partial constriction of the selectivity filter. Recent studies have identified a number of phenotypic differences in the inactivation properties of different Kv channels, including different sensitivities to elevation of extracellular K⁺ concentration, and different state dependencies of inactivation. We have demonstrated that an alternatively spliced short form of Kv1.5, resulting in disruption of the T1 domain, exhibits a shift in the state dependence of inactivation in this channel, and in the current study we have examined this further to contrast the properties of inactivation from open versus closed states. In a TEA⁺-sensitive mutant of Kv1.5 (Kv1.5 R487T), 10 m m extracellular TEA⁺ inhibits inactivation in both full-length and T1-deleted channels, but does not inhibit closed-state inactivation in T1-deleted channel forms. Similarly, substitution of K⁺ and Na⁺ with Cs⁺ ions in the recording medium inhibits inactivation of both full-length and T1-deleted channel forms, but fails to inhibit closed-state inactivation of T1-deleted channels. Collectively, these data distinguish between open-state and closed-state inactivation, and suggest the presence of multiple possible mechanisms of inactivation coexisting in Kv1 channels.     

Heterogeneous expression of KChIP2 isoforms in the ferret heart

Kv4 channels are believed to underlie the rapidly recovering cardiac transient outward current ( I _to) phenotype. However, heterologously expressed Kv4 channels fail to fully reconstitute the native current. Kv channel interacting proteins (KChIPs) have been shown to modulate Kv4 channel function. To determine the potential involvement of KChIPs in the rapidly recovering I _to, we cloned three KChIP2 isoforms (designated fKChIP2, 2a and 2b) from the ferret heart. Based upon immunoblot data suggesting the presence of a potential endogenous KChIP-like protein in HEK 293, CHO and COS cells but absence in Xenopus oocytes, we coexpressed Kv4.3 and the fKChIP2 isoforms in Xenopus oocytes. Functional analysis showed that while all fKChIP2 isoforms produced a fourfold acceleration of recovery kinetics compared to Kv4.3 expressed alone, only fKChIP2a produced large depolarizing shifts in the V _1/2 of steady-state activation and inactivation as seen for the native rapidly recovering I _to. Analysis of RNA and protein expression of the three fKChIP2 isoforms in ferret ventricles showed that fKChIP2b was most abundant and was expressed in a gradient paralleling the rapidly recovering I _to distribution. Ferret KChIP2 and 2a were expressed at very low levels. The ventricular expression distribution suggests that fKChIP2 isoforms are involved in modulation of the rapidly recovering I _to; however, additional regulatory factors are also likely to be involved in generating the native current.     

Regulation of cardiac shal-related potassium channel Kv 4.3 by serum- and glucocorticoid-inducible kinase isoforms in Xenopus oocytes

The human cardiac transient outward potassium current I_to is formed by co-assembly of voltage-dependent K⁺ channel (Kv 4.3) pore-forming -subunits with differently spliced K channel interacting protein (KChIP) accessory proteins. I_to is of considerable importance for the normal course of the cardiac ventricular action potential. The present study was performed to determine whether isoforms of the serum- and glucocorticoid-inducible kinase (SGK) family influence Kv 4.3/KChIP2b channel activity in the Xenopus laevis heterologous expression system. Co-expression of SGK1, but not of SGK2 or SGK3, increased Kv 4.3/KChIP2b channel currents. The up-regulation of the current was not due to changes in the activation curve or changes of channel inactivation. The currents in oocytes expressing Kv 4.3 alone were smaller than those in Kv 4.3/KChIP2b expressing oocytes, but were still stimulated by SGK1. The effect of wild-type SGK1 was mimicked by constitutively active SGK1 (SGK1 S422D) but not by an inactive mutant (SGK1 K127N). The current amplitude increase mediated by SGK1 was not dependent on NEDD4.2 or RAB5, nor did it reflect increased cell surface expression. In conclusion, SGK1 stimulates Kv 4.3 potassium channels expressed in Xenopus oocytes by a novel mechanism distinct from the known NEDD4.2-dependent pathway.     

Temperature and redox state dependence of native Kv2.1 currents in rat pancreatic β-cells

In pancreatic β-cells, voltage-dependent K⁺ (Kv) channels repolarise glucose-stimulated action potentials. Kv channels are therefore negative regulators of Ca²⁺ entry and insulin secretion. We have recently demonstrated that Kv2.1 mediates the majority of β-cell voltage-dependent outward K⁺ current and now investigate the function of native β-cell Kv2.1 channels at near-physiological temperatures (32-35 °C). While β-cell voltage-dependent outward K⁺ currents inactivated little at room temperature, both fast-inactivation (111.5 ± 14.3 ms) and slow-inactivation (1.21 ± 0.12 s) was observed at 32-35 °C. Kv2.1 mediates the fast-inactivating current observed at 32-35 °C, since it could be selectively ablated by expression of a dominant-negative Kv2.1 construct (Kv2.1N). The surprising ability of Kv2.1N to selectively remove the fast-inactivating component, together with its sensitivity to tetraethylammonium (TEA), demonstrate that this component is not mediated by the classically fast-inactivating and TEA-resistant channels such as Kv1.4 and 4.2. Increasing the intracellular redox state by elevating the cytosolic NADPH/NADP⁺ ratio from 1/10 to 10/1 increased the rates of both fast- and slow-inactivation. In addition, increasing the intracellular redox state also increased the relative contribution of the fast-inactivation component from 38.8 ± 2.1 % to 55.9 ± 1.8 %. The present study suggests that, in β-cells, Kv2.1 channels mediate a fast-inactivating K⁺ current at physiological temperatures and may be regulated by the metabolic generation of NADPH.     

Expression and function of dipeptidyl-aminopeptidase-like protein 6 as a putative β-subunit of human cardiac transient outward current encoded by Kv4.3

Dipeptidyl-aminopeptidase-like protein 6 (DPPX) was recently shown in the brain to modulate the kinetics of transient A-type currents by accelerating inactivation and recovery from inactivation. Since the kinetics of human cardiac transient outward current ( I _to) are not mimicked by coexpression of the α-subunit Kv4.3 with its known β-subunit KChIP2, we have tested the hypothesis that DPPX may serve as an additional β-subunit in the human heart. With quantitative real-time RT-PCR strong mRNA expression of DPPX was detected in human ventricles and was verified at the protein level in human but not in rat heart by a DPPX-specific antibody. Co-expression of DPPX with Kv4.3 in Chinese hamster ovary cells produced I _to-like currents, but compared with expression of KChIP2a and Kv4.3, the time constant of inactivation was faster, the potential of half-maximum steady-state inactivation was more negative and recovery from inactivation was delayed. Co-expression of DPPX in addition to Kv4.3 and KChIP2a produced similar current kinetics as in human ventricular myocytes. We therefore propose that DPPX is an essential component of the native cardiac I _to channel complex in human heart.     

Down regulation of Kv3.4 channels by chronic hypoxia increases acute oxygen sensitivity in rabbit carotid body

The carotid body (CB) chemoreceptors participate in the ventilatory responses to acute and chronic hypoxia (CH). Arterial hypoxaemia increases breathing within seconds, and CB chemoreceptors are the principal contributors to this reflex hyperventilatory response. Acute hypoxia induces depolarization of CB chemoreceptors by inhibiting certain K⁺ channels, but the role of these channels in CH, as in high-altitude acclimatization, is less known. Here we explored the effects of prolonged (24–48 h) hypoxic exposure of rabbit CB chemoreceptor cells in primary cultures on the voltage-dependent K⁺ currents and on their response to acute hypoxia. We found that CH induces a decrease in the amplitude of outward K⁺ currents due to a reduction in a fast-inactivating BDS- and highly TEA-sensitive component of the current. In spite of this effect, acute hypoxic inhibition of K⁺ currents is increased in CH cultures, as well as hypoxia-induced depolarization. These data suggest that downregulation of this component (that does not contribute to the oxygen-sensitive K⁺ current ( I _KO2)) participates in the hypoxic sensitization. Pharmacological, immunocytochemical and quantitative PCR (qPCR) experiments demonstrate that CH-induced decrease in outward K⁺ currents is due to a downregulation of the expression of Kv3.4 channels. Taken together, our results suggest that CH sensitization in rabbit CB could be achieved by an increase in the relative contribution of I _KO2 to the outward K⁺ current as a consequence of the decreased expression of the oxygen-insensitive component of the current. We conclude that acute and chronic hypoxia can exert their effects acting on different molecular targets.     

Developmental changes in potassium currents at the rat calyx of Held presynaptic terminal

During early postnatal development, the calyx of Held synapse in the auditory brainstem of rodents undergoes a variety of morphological and functional changes. Among ionic channels expressed in the calyx, voltage-dependent K⁺ channels regulate transmitter release by repolarizing the nerve terminal. Here we asked whether voltage-dependent K⁺ channels in calyceal terminals undergo developmental changes, and whether they contribute to functional maturation of this auditory synapse. From postnatal day (P) 7 to P14, K⁺ currents became larger and faster in activation kinetics, but did not change any further to P21. Likewise, presynaptic action potentials became shorter in duration from P7 to P14 and remained stable thereafter. The density of presynaptic K⁺ currents, assessed from excised patch recording and whole-cell recordings with reduced [K⁺]_i, increased by 2–3-fold during the second postnatal week. Pharmacological isolation of K⁺ current subtypes using tetraethylammonium (1 m m ) and margatoxin (10 n m ) revealed that the density of Kv3 and Kv1 currents underwent a parallel increase, and their activation kinetics became accelerated by 2–3-fold. In contrast, BK currents, isolated using iberiotoxin (100 n m ), showed no significant change during the second postnatal week. Pharmacological block of Kv3 or Kv1 channels at P7 and P14 calyceal terminals indicated that the developmental changes of Kv3 channels contribute to the establishment of reliable action potential generation at high frequency, whereas those of Kv1 channels contribute to stabilizing the nerve terminal. We conclude that developmental changes in K⁺ currents in the nerve terminal contribute to maturation of high-fidelity fast synaptic transmission at this auditory relay synapse.     

Kv2 subunits underlie slowly inactivating potassium current in rat neocortical pyramidal neurons

We determined the expression of Kv2 channel subunits in rat somatosensory and motor cortex and tested for the contributions of Kv2 subunits to slowly inactivating K⁺ currents in supragranular pyramidal neurons. Single cell RT-PCR showed that virtually all pyramidal cells expressed Kv2.1 mRNA and ∼80% expressed Kv2.2 mRNA. Immunocytochemistry revealed striking differences in the distribution of Kv2.1 and Kv2.2 subunits. Kv2.1 subunits were clustered and located on somata and proximal dendrites of all pyramidal cells. Kv2.2 subunits were primarily distributed on large apical dendrites of a subset of pyramidal cells from deep layers. We used two methods for isolating currents through Kv2 channels after excluding contributions from Kv1 subunits: intracellular diffusion of Kv2.1 antibodies through the recording pipette and extracellular application of rStromatoxin-1 (ScTx). The Kv2.1 antibody specifically blocked the slowly inactivating K⁺ current by 25–50% (at 8 min), demonstrating that Kv2.1 subunits underlie much of this current in neocortical pyramidal neurons. ScTx (300 n m ) also inhibited ∼40% of the slowly inactivating K⁺ current. We observed occlusion between the actions of Kv2.1 antibody and ScTx. In addition, Kv2.1 antibody- and ScTx-sensitive currents demonstrated similar recovery from inactivation and voltage dependence and kinetics of activation and inactivation. These data indicate that both agents targeted the same channels. Considering the localization of Kv2.1 and 2.2 subunits, currents from truncated dissociated cells are probably dominated by Kv2.1 subunits. Compared with Kv2.1 currents in expression systems, the Kv2.1 current in neocortical pyramidal cells activated and inactivated at relatively negative potentials and was very sensitive to holding potential.     

Ileitis modulates potassium and sodium currents in guinea pig dorsal root ganglia sensory neurons

Intestinal inflammation induces hyperexcitability of dorsal root ganglia sensory neurons, which has been implicated in increased pain sensation. This study examined whether alteration of sodium (Na⁺) and/ or potassium (K⁺) currents underlies this hyperexcitability. Ileitis was induced in guinea pig ileum with trinitrobenzene sulphonic acid (TBNS) and dorsal root ganglion neurons innervating the site of inflammation were identified by Fast Blue or DiI fluorescence labelling. Whole cell recordings were made from acutely dissociated small-sized neurons at 7–10 days. Neurons exhibited transient A-type and sustained outward rectifier K⁺ currents. Compared to control, both A-type and sustained K⁺ current densities were significantly reduced (42 and 34 %, respectively;   P < 0.05  ) in labelled neurons from the inflamed intestine but not in non-labelled neurons. A-type current voltage dependence of inactivation was negatively shifted in labelled inflamed intestine neurons. Neurons also exhibited tetrodotoxin-sensitive and resistant Na⁺ currents. Tetrodotoxin-resistant sodium currents were increased by 37 % in labelled neurons from the inflamed intestine compared to control (   P < 0.01  ), whereas unlabelled neurons were unaffected. The activation and inactivation curves of these currents were unchanged by inflammation. These data suggest ileitis increases excitability of intestinal sensory neurons by modulating multiple ionic channels. The lack of effect in non-labelled neurons suggests signalling originated at the nerve terminal rather than through circulating mediators and, given that Na⁺ currents are enhanced whereas K⁺ currents are suppressed, one or more signalling pathways may be involved.     

Properties and molecular basis of the mouse urinary bladder voltage-gated K+ current

Potassium channels play an important role in controlling the excitability of urinary bladder smooth muscle (UBSM). Here we describe the biophysical, pharmacological and molecular properties of the mouse UBSM voltage-gated K⁺ current ( I _{K ( V)}). The I _{K ( V)} activated, deactivated and inactivated slowly with time constants of 29.9 ms at +30 mV, 131 ms at −40 mV and 3.4 s at +20 mV. The midpoints of steady-state activation and inactivation curves were 1.1 mV and −61.4 mV, respectively. These properties suggest that I _{K ( V)} plays a role in regulating the resting membrane potential and contributes to the repolarization and after-hyperpolarization phases of action potentials. The I _{K ( V)} was blocked by tetraethylammonium ions with an IC₅₀ of 5.2 m m and was unaffected by 1 m m 4-aminopyridine. RT-PCR for voltage-gated K⁺ channel (K_V) subunits revealed the expression of Kv2.1, Kv5.1, Kv6.1, Kv6.2 and Kv6.3 in isolated UBSM myocytes. A comparison of the biophysical properties of UBSM I _{K ( V)} with those reported for Kv2.1 and Kv5.1 and/or Kv6 heteromultimeric channels demonstrated a marked similarity. We propose that heteromultimeric channel complexes composed of Kv2.1 and Kv5.1 and/or Kv6 subunits form the molecular basis of the mouse UBSM I _{K ( V)}.     

Regulation of apoptotic potassium currents by coordinated zinc-dependent signalling

Oxidant-liberated intracellular Zn²⁺ regulates neuronal apoptosis via an exocytotic membrane insertion of Kv2.1-encoded ion channels, resulting in an enhancement of voltage-gated K⁺ currents and a loss of intracellular K⁺ that is necessary for caspase-mediated proteolysis. In the present study we show that an N-terminal tyrosine of Kv2.1 (Y124), which is a known target of Src kinase, is critical for the apoptotic current surge. Moreover, we demonstrate that Y124 works in concert with a C-terminal serine (S800) target of p38 mitogen-activated protein kinase (MAPK) to regulate Kv2.1-mediated current enhancement. While Zn²⁺ was previously shown to activate p38, we show here that this metal inhibits cytoplasmic protein tyrosine phosphatase ɛ (Cyt-PTPɛ), which specifically targets Y124. Importantly, a point mutation of Y124 to a non-phosphorylatable residue or over-expression of Cyt-PTPɛ protects cells from injury. Kv2.1-encoded channels thus regulate neuronal survival by providing a converging input for two Zn²⁺-dependent signal transduction cascades.