Molecular basis for different pore properties of potassium channels from the rat brain Kv1 gene family.

Members of the rat brain Kv1 family of cloned potassium channels are structurally highly homologous, but have diverse conductance and pharmacological characteristics. Here we present data on the effects of mutating residues K533 in the P-region and H471 in the S4-S5 linker of Kv1.4 to their equivalent residues in Kv1.1 and Kv1.6 on single-channel conductance and sensitivity to external tetraethylammonium cations (TEA+) and internal Mg2+. Exchange of residue K533 for its equivalent residue (Y) in Kv1.1 and Kv1.6 increased the single-channel conductance at both negative and positive potentials. This mutation is known to reduce the IC50 for external TEA+ from > 100 mM to 0.6 mM, almost identical to that for Kv1.1 (0.53 mM). We have now found that the additional exchange of residue H471 for the equivalent residue (K) in Kv1.6 increased the IC50 for external TEA+ from 0.6 mM (Kv1.4K533Y) to 2.39 mM; this is very close to that for wild-type Kv1.6 channels (2.84 mM). The mutation H471K alone was ineffective. We thus provide evidence that the S4-S5 linker does contribute to the channel's inner-pore region. Data on the block of Kv1 channels by internal Mg2+ indicate that while the binding site is probably situated within the deep-pore region, its exact location may be channel specific.

Frequency-dependent inactivation of mammalian A-type K+ channel KV1.4 regulated by Ca2+/calmodulin-dependent protein kinase.

Ca2+/calmodulin dependent protein kinase (CaMKII) and protein phosphatase 2B (calcineurin) are key enzymes in the regulation of synaptic strength, controlling the phosphorylation status of pre- and postsynaptic target proteins. Here, we show that the inactivation gating of the Shaker-related fast-inactivating KV channel, Kv1.4 is controlled by CaMKII and the calcineurin/inhibitor-1 protein phosphatase cascade. CaMKII phosphorylation of an amino-terminal residue of KV1.4 leads to slowing of inactivation gating and accelerated recovery from N-type inactivated states. In contrast, dephosphorylation of this residue induces a fast inactivating mode of KV1.4 with time constants of inactivation 5 to 10 times faster compared with the CaMKII-phosphorylated form. Dephosphorylated KV1.4 channels also display slowed and partial recovery from inactivation with increased trapping of KV1.4 channels in long-absorbing C-type inactivated states. In consequence, dephosphorylated KV1.4 displays a markedly increased tendency to undergo cumulative inactivation during repetitive stimulation. The balance between phosphorylated and dephosphorylated KV1.4 channels is regulated by changes in intracellular Ca2+ concentration rendering KV1.4 inactivation gating Ca2+-sensitive. The reciprocal CaMKII and calcineurin regulation of cumulative inactivation of presynaptic KV1.4 may provide a novel mechanism to regulate the critical frequency for presynaptic spike broadening and induction of synaptic plasticity.

Functional differences of a Kv2.1 channel and a Kv2.1/Kv1.2S4-chimera are confined to a concerted voltage shift of various gating parameters.

When expressed in Xenopus oocytes, the voltage-dependent K+ channels Kv1.2 and Kv2.1 have similar steady state parameters of activation but the kinetics of activation is significantly faster in the Kv1.2 channels. Activation results from intramolecular arrangements which start with the movement of the voltage sensor and end with the opening of the pore. The S4-segment and the H5-loop comprise at least part of the respective involved structural elements. The molecular mechanism of coupling between sensing of voltage and opening of the pore is less well understood. We have measured whole cell and single channel ionic currents in the rapidly activating Kv1.2 channel of the rat, the slowly activating Kv2.1 channel of the human, and in an S4-chimera Kv2.1/Kv1.2S4. With respect to the Kv2.1 channel, steady state activation and steady state C-type inactivation of the chimeric channel are shifted by more than 50 mV in the depolarizing direction. The property of rapid activation in Kv1.2 channels was not transferred to the Kv2.1 channels with the transplanted S4-region. Instead, the kinetics of activation, deactivation, and recovery from C-type inactivation as well as the voltage sensitivity of the 4-aminopyridine block are similar to the corresponding processes in Kv2.1 channels if they are related to the steady state activation and inactivation, respectively. The unitary current and the mean open time of single channel openings of the S4-chimeric channels resemble the respective values of Kv2.1 channels. It is concluded that the insertion of the S4-segment of Kv1.2 channels into Kv2.1 channels modifies the gating at the early steps of activation leaving all properties associated with the open state(s) of the Kv2.1 channels unaffected.

Cooperativity of phosphatidylinositol transfer protein and phospholipase D in secretory vesicle formation from the TGN--phosphoinositides as a common denominator?

Phosphatidylinositol transfer protein (PITP) and phospholipase D (PLD) stimulate the formation of constitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans-Golgi network (TGN) in a cell-free system. The stimulatory effects of PITP and PLD are additive. Stimulation by either PITP or PLD is blocked by geneticin, a member of the aminoglycoside antibiotics known to bind to phosphoinositides. Since the PLD we used is insensitive to geneticin, our results suggest that phosphoinositides promote secretory vesicle formation as downstream effectors of both PITP and PLD, possibly via the recruitment of proteins mediating membrane budding and fission.

Mechanism of action of the epileptogenic drug pentylenetetrazol on a cloned neuronal potassium channel.

The action of the epileptogenic agent pentylenetetrazol (PTZ) on a cloned potassium channel of the rat brain was studied. The Kv1.1 channel was expressed in oocytes of Xenopus laevis and potassium currents were investigated in outside-out and inside-out membrane patches. The results show that PTZ increased the multi-channel potassium currents at strongly negative potentials and decreased them at potentials positive to -35 mV both in outside-out and inside-out membrane patches. The extent and manner of PTZ action, the concentration dependence as well as the onset and time course of the PTZ effect were the same both in outside-out and inside-out membrane patches. The single-channel potassium currents showed an increase in open probability and frequency of opening and a decrease in close time at -50 mV and vice versa at 0 mV with application of PTZ. The amplitude of single-channel current, the open time and the latency to the first channel opening remained almost unchanged under PTZ. The results indicate that PTZ acts via the cell membrane and influences the membrane-associated part of the potassium channel. Thereby, PTZ accelerates the transition from the inactivated to the open state of the channel at strongly negative potentials and reduces it at slightly negative and positive potentials. This mechanism may be the basis for a gate function which is in favour of the development of epileptic discharges.

Gating and conductance properties of a human delayed rectifier K+ channel expressed in frog oocytes.

1. The human delayed rectifier K+ channel h-DRK1, a homologue to the DRK1 channel in the rat, was expressed in Xenopus oocytes. Single-channel currents were measured in micropatches; macroscopic currents were measured either in macropatches, giant patches, or whole oocytes. 2. Macroscopic currents activated at -20 mV and more positive. The instantaneous current-voltage relationship rectified outwardly to a higher degree than predicted by the Goldman-Hodgkin-Katz equation. 3. With the giant patch technique, ionic and putative on- and off-gating currents were recorded simultaneously. The large ratio of the moved gating charges to the amplitude of the ionic current indicated that less than 1% of the gating channels actually opened. 4. The single-channel conductance between 0 and +80 mV was calculated to be 9.4 pS. The channels opened with sublevels which appeared either independently of the fully open level as separate openings, in conjunction with the opening to and closing from the fully open level, or by starting from and ending at the fully open level. 5. The channels opened with two voltage-independent open time constants in the range 1-10 ms (filter 1 kHz). The burst open probability was fitted monoexponentially with time constants in the range of tens of milliseconds. 6. Assuming a sequential Markovian model with four independent voltage-controlled transitions, fit of the steady-state open probability of macroscopic currents showed two components of activation differing in their half-maximal value. 7. The fit of time courses of cumulative first latency and ensemble-averaged currents in single-channel patches suggested that even a single channel may operate with the two different components of activation. 8. It is concluded that h-DRK1 channels considerably rectify in an outward direction and that an apparently flat voltage dependence of activation may be explained by the overlap of two different components.

Inactivation properties of voltage-gated K+ channels altered by presence of beta-subunit.

Structural and functional diversity of voltage-gated Kv1-type potassium channels in rat brain is enhanced by the association of two different types of subunits, the membrane-bound, poreforming alpha-subunits and a peripheral beta-subunit. We have cloned a beta-subunit (Kv beta 1) that is specifically expressed in the rat nervous system. Association of Kv beta 1 with alpha-subunits confers rapid A-type inactivation on non-inactivating Kv1 channels (delayed rectifiers) in expression systems in vitro. This effect is mediated by an inactivating ball domain in the Kv beta 1 amino terminus.

The inactivation behaviour of voltage-gated K-channels may be determined by association of alpha- and beta-subunits.

Voltage-gated K-channels of the Shaker related subfamily have two subunits, membrane integrated alpha- and peripheral beta-subunits. alpha-Subunits may assemble as tetramers and form in in vitro expression systems functional K-channels. beta-Subunits cannot from channels by themselves. Like for alpha-subunits, the rat nervous system apparently expresses a family of beta-subunit proteins. We have demonstrated that one rat K-channel beta-subunit, Kv beta 1, contains an inactivating domain. Upon association of alpha- and Kv beta 1-subunits, delayed-rectifier type K-channels are converted to rapidly inactivating A-type K-channels. The beta-subunit inactivation domain acts via a ball and chain type mechanism previously proposed for N-type inactivation of alpha-subunits. The association of alpha- and beta-subunits endows the nervous system with an unprecedented flexibility and diversity of K-channels which may play an important role in the regulation of nervous excitability.

A site accessible to extracellular TEA+ and K+ influences intracellular Mg2+ block of cloned potassium channels.

The members of the RCK family of cloned voltage-dependent K+ channels are quite homologous in primary structure, but they are highly diverse in functional properties. RCK4 channels differ from RCK1 and RCK2 channels in inactivation and permeation properties, the sensitivity to external TEA, and to current modulation by external K+ ions. Here we show several other interesting differences: While RCK1 and RCK2 are blocked in a voltage and concentration dependent manner by internal Mg2+ ions, RCK4 is only weakly blocked at very high potentials. The single-channel current-voltage relations of RCK4 are rather linear while RCK2 exhibits an inwardly rectifying single-channel current in symmetrical K+ solutions. The deactivation of the channels, measured by tail current protocols, is faster in RCK4 by a factor of two compared with RCK2. In a search for the structural motif responsible for these differences, point mutants creating homology between RCK2 and RCK4 in the pore region were tested. The single-point mutant K533Y in the background of RCK4 conferred the properties of Mg2+ block, tail current kinetics, and inward ion permeation of RCK2 to RCK4. This mutant was previously shown to be responsible for the alterations in external TEA sensitivity and channel regulation by external K+ ions. Thus, this residue is expected to be located at the external side of the pore entrance. The data are consistent with the idea that the mutation alters the channel occupancy by K+ and thereby indirectly affects internal Mg2+ block and channel closing.

Cloning and characterization of a human delayed rectifier potassium channel gene.

A human genomic DNA library was screened for sequences homologues to the rat delayed rectifier Kv 2.1 (DRK1) K+ channel cDNA. Three phages were isolated which hybridized to Kv 2.1 cDNA probes. Alignment of the human genomic DNA sequence with the rat cDNA sequence indicated that the open reading frame (ORF) is interrupted by a large intervening sequence, that separates exons encoding the membrane spanning core region of the K+ channel polypeptide. The Kv 2.1 gene occurs once in the human genome and has been mapped to chromosome 20. The human, mouse and rat Kv 2.1 proteins have been highly conserved, showing only a few substitutions outside of the membrane spanning domains in the amino- and carboxy-terminal cytoplasmic domains. Nevertheless, expression of human DRK1 channels in Xenopus oocytes showed that mouse, rat and human Kv 2.1 channels have distinct pharmacological and electrophysiological properties. The observed differences in activation, voltage-dependence, 4-aminopyridine sensitivity and single-channel conductance have to be attributed to amino acid substitutions in the amino-and/or carboxy-terminal cytoplasmic domains. Obviously, these domains of Kv 2.1 channels influence biophysical K+ channel properties, which are thought to be determined solely by the membrane spanning core domain of potassium channels.

Extracellular K+ specifically modulates a rat brain K+ channel.

Extracellular potassium concentration is actively maintained within narrow limits in all higher organisms. Slight variations in extracellular potassium levels can induce major alterations of essential physiological functions in excitable tissues. Here we describe that superfusion of cultured rat hippocampal neurones with potassium-free medium leads to a decrease of a specific outward potassium current, probably carried by RCK4-type channels (RCK4 are potassium channels found in rat brain). This is confirmed by heterologous expression of these channels in Xenopus oocytes. In this system, variations of extracellular potassium in the physiological concentration range induce significant differences in current amplitude. Moreover, the current is completely suppressed in the absence of extracellular potassium. The potassium dependence of macroscopic conductance in RCK4 channels was related by site-directed mutagenesis to that lysine residue in the extracellular loop between the transmembrane segments S5 and S6 of RCK4 protein that confers resistance to extracellular blockage by tetraethylammonium. It is shown that extracellular potassium affects the number of available RCK4 channels, but not the single-channel conductance, the mean open time, or the gating charge displacement upon depolarization.