大鼠背根神经节神经元上失活 BK 通道特性的研究

大电导的电压和 Ca²⁺ 激活的 K⁺ 通道 (BK 通道 ) 在哺乳动物的组织中广泛表达,起着多种多样的作用 . 目前只有少数组织中 BK 通道的性质被深入地研究,而且鲜见有失活的 BK 通道 (BK_i) 的报道,尤其是在神经元中 . 发现在大鼠小直径的背根神经节 (DRG) 神经元中,普遍存在失活的 BK 通道 . 失活的 BK 电流成分是 Ca²⁺敏感的,可以被大电导的 BK 通道特异阻断剂 ChTX 所阻断,而且木瓜蛋白酶可以从胞外改变通道失活的特性 .     

N-type Inactivation in the Mammalian Shaker K+ Channel Kv1.4

Mammalian voltage-gated K⁺ channels are oligomeric proteins, some of which may be composed in vivo of subunits derived from several similar genes. We have studied N-type inactivation in the rapidly inactivating Kv1.4 channel and, in specific, heteromultimers of this gene product with Kv1.5 noninactivating subunits. Heteromultimeric channels were analyzed for the stoichiometry of Kv1.4:Kv1.5 subunits by observing shifts in the midpoints of steady-state availability from that of homomultimeric channels. This analysis was employed to examine inactivation of heteromultimeric channels expressed in Xenopus oocytes using two model systems: by expression of a Kv1.4–Kv1.5 tandem fusion construct and by coexpression of native Kv1.4 and Kv1.5 channels across a wide relative concentration range of microinjected mRNA. Additionally, inactivation was examined in coexpression experiments of N-terminal deletion mutants of Kv1.4. We found that (i) a single inactivating subunit conferred inactivation in all hetero-multimers studied; (ii) the rate of inactivation could not be distinguished in channels containing two inactivating subunits from those containing one inactivating subunit; and (iii) large deletions in the linker region between the N-terminal inactivation region and the first membrane-spanning domain had no effect on the rate of inactivation. These data confirm the importance of the proximal N-terminal region in the inactivation of mammalian Kv1.4 channels, and suggest that the inactivation particle remains in close proximity to the permeation pathway even when the channel is in the open state. Received: 24 August 1995/Revised: 7 February 1996     

Multiple structural domains contribute to voltage-dependent inactivation of rat brain alpha(1E) calcium channels.

We have investigated the molecular determinants that mediate the differences in voltage-dependent inactivation properties between rapidly inactivating (R-type) alpha(1E) and noninactivating (L-type) alpha(1C) calcium channels. When coexpressed in human embryonic kidney cells with ancillary beta(1b) and alpha(2)-delta subunits, the wild type channels exhibit dramatically different inactivation properties; the half-inactivation potential of alpha(1E) is 45 mV more negative than that observed with alpha(1C), and during a 150-ms test depolarization, alpha(1E) undergoes 65% inactivation compared with only about 15% for alpha(1C). To define the structural determinants that govern these intrinsic differences, we have created a series of chimeric calcium channel alpha(1) subunits that combine the major structural domains of the two wild type channels, and we investigated their voltage-dependent inactivation properties. Each of the four transmembrane domains significantly affected the half-inactivation potential, with domains II and III being most critical. In particular, substitution of alpha(1C) sequence in domains II or III with that of alpha(1E) resulted in 25-mV negative shifts in half-inactivation potential. Similarly, the differences in inactivation rate were predominantly governed by transmembrane domains II and III and to some extent by domain IV. Thus, voltage-dependent inactivation of alpha(1E) channels is a complex process that involves multiple structural domains and possibly a global conformational change in the channel protein.     

Cardiac Na currents and the inactivating, reopening, and waiting properties of single cardiac Na channels

Tetrodotoxin (TTX)-sensitive Na currents were examined in single dissociated ventricular myocytes from neonatal rats. Single channel and whole cell currents were measured using the patch-clamp method. The channel density was calculated as 2/micron 2, which agreed with our usual finding of four channels per membrane patch. At 20 degrees C, the single channel conductance was 20 pS. The open time distributions were fit by a single-exponential function with a mean open time of approximately 1.0 ms at membrane potentials from -60 to -40 mV. Averaged single channel and whole cell currents were similar when scaled and showed both fast and slow rates of inactivation. The inactivation and activation gating shifted quickly to hyperpolarized potentials for channels in cell-attached as well as excised patches, whereas a much slower shift occurred in whole cells. Slowly inactivating currents were present in both whole cell and single channel current measurements at potentials as positive as -40 mV. In whole cell measurements, the potential range could be extended, and slow inactivation was present at potentials as positive as -10 mV. The curves relating steady state activation and inactivation to membrane potential had very little overlap, and slow inactivation occurred at potentials that were positive to the overlap. Slow inactivation is in this way distinguishable from the overlap or window current, and the slowly inactivating current may contribute to the plateau of the rat cardiac action potential. On rare occasions, a second set of Na channels having a smaller unit conductance and briefer duration was observed. However, a separate set of threshold channels, as described by Gilly and Armstrong (1984. Nature [Lond.]. 309:448), was not found. For the commonly observed Na channels, the number of openings in some samples far exceeded the number of channels per patch and the latencies to first opening or waiting times were not sufficiently dispersed to account for the slowly inactivating currents: the slow inactivation was produced by channel reopening. A general model was developed to predict the number of openings in each sample. Models in which the number of openings per sample was due to a dispersion of waiting times combined with a rapid transition from an open to an absorbing inactivated state were unsatisfactory and a model that was more consistent with the results was identified.     

Characterization of voltage-and Ca2+-activated K+ channels in rat dorsal root ganglion neurons

Auxiliary beta-subunits associated with pore-forming Slo1 alpha-subunits play an essential role in regulating functional properties of large-conductance, voltage- and Ca(2+)-activated K(+) channels commonly termed BK channels. Even though both noninactivating and inactivating BK channels are thought to be regulated by beta-subunits (beta1, beta2, beta3, or beta4), the molecular determinants underlying inactivating BK channels in native cells have not been extensively demonstrated. In this study, rbeta2 (but not rbeta3-subunit) was identified as a molecular component in rat lumbar L4-6 dorsal root ganglia (DRG) by RT-PCR responsible for inactivating large-conductance Ca(2+)-dependent K(+) currents (BK(i) currents) in small sensory neurons. The properties of native BK(i) currents obtained from both whole-cell and inside-out patches are very similar to inactivating BK channels produced by co-expressing mSlo1 alpha- and hbeta2-subunits in Xenopus oocytes. Intracellular application of 0.5 mg/ml trypsin removes inactivation of BK(i) channels, and the specific blockers of BK channels, charybdotoxin (ChTX) and iberiotoxin (IbTX), inhibit these BK(i) currents. Single BK(i) channel currents derived from inside-out patches revealed that one BK(i) channel contained three rbeta2-subunits (on average), with a single-channel conductance about 217 pS under 160 K(+) symmetrical recording conditions. Blockade of BK(i) channels by 100 nM IbTX augmented firing frequency, broadened action potential waveform and reduced after-hyperpolarization. We propose that the BK(i) channels in small diameter DRG sensory neurons might play an important role in regulating nociceptive input to the central nervous system (CNS).     

Steady-state and closed-state inactivation properties of inactivating BK channels

Calcium-dependent potassium (BK-type) Ca2+ and voltage-dependent K+ channels in chromaffin cells exhibit an inactivation that probably arises from coassembly of Slo1 alpha subunits with auxiliary beta subunits. One goal of this work was to determine whether the Ca2+ dependence of inactivation arises from any mechanism other than coupling of inactivation to the Ca2+ dependence of activation. Steady-state inactivation and the onset of inactivation were studied in inside-out patches and whole-cell recordings from rat adrenal chromaffin cells with parallel experiments on inactivating BK channels resulting from cloned alpha + beta2 subunits. In both cases, steady-state inactivation was shifted to more negative potentials by increases in submembrane [Ca2+] from 1 to 60 microM. At 10 and 60 microM Ca2+, the maximal channel availability at negative potentials was similar despite a shift in the voltage of half availability, suggesting there is no strictly Ca2+-dependent inactivation. In contrast, in the absence of Ca2+, depolarization to potentials positive to +20 mV induces channel inactivation. Thus, voltage-dependent, but not solely Ca2+-dependent, kinetic steps are required for inactivation to occur. Finally, under some conditions, BK channels are shown to inactivate as readily from closed states as from open states, indicative that a key conformational change required for inactivation precedes channel opening.     

Distribution of single-channel conductances in cultured rat hippocampal neurons

1. The nonhomogeneous spatial distribution of ionic channels in neurons has been implied from intracellular recordings at somatic and dendritic locations. These reports indicate that Na- and Ca-dependent regenerative currents are distributed differently throughout the neuron. Although a variety of K conductances and a noninactivating Na conductance have been described in intracellular studies, little is known about the spatial distribution of inward and outward currents throughout different regions of the neuron. 2. We recorded from cell-attached patches from cultured hippocampal cells from 1-day-old rats. The cells were cultured for 3-21 days. The spatial distribution of a variety of ionic channels was determined by comparing the conductances from somatic and dendritic membranes. Single-channel currents obtained from cell-attached patches were identified by the time course of ensemble (averaged) responses, voltage dependence, and the effect of channel blocking agents. 3. We consistently observed that only the rapidly inactivating inward current was localized to the soma. The other channel types that we studied, including an inward noninactivating, delayed rectifier and transient A-type currents, were observed in both the somatic and dendritic regions. 4. We suggest that the distribution of ionic conductances that we have observed may be functional in limiting excitability during development of neurons.     

Tyrosine phosphorylation of the inactivating peptide of the shaker B potassium channel: a structural-functional correlate

A synthetic peptide patterned after the sequence of the inactivating "ball" domain of the Shaker B K(+) channel restores fast (N-type) inactivation in mutant deletion channels lacking their constitutive ball domains, as well as in K(+) channels that do not normally inactivate. We now report on the effect of phosphorylation at a single tyrosine in position 8 of the inactivating peptide both on its ability to restore fast channel inactivation in deletion mutant channels and on the conformation adopted by the phosphorylated peptide when challenged by anionic lipid vesicles, a model target mimicking features of the inactivation site in the channel protein. We find that the inactivating peptide phosphorylated at Y8 behaves functionally as well as structurally as the noninactivating mutant carrying the mutation L7E. Moreover, it is observed that the inactivating peptide can be phosphorylated by the Src tyrosine kinase either as a free peptide in solution or when forming part of the membrane-bound protein channel as the constitutive inactivating domain. These findings suggest that tyrosine phosphorylation-dephosphorylation of this inactivating ball domain could be of physiological relevance to rapidly interconvert fast-inactivating channels into delayed rectifiers and vice versa.     

Bupivacaine blocks N-type inactivating Kv channels in the open state: no allosteric effect on inactivation kinetics

Local anesthetics bind to ion channels in a state-dependent manner. For noninactivating voltage-gated K channels the binding mainly occurs in the open state, while for voltage-gated inactivating Na channels it is assumed to occur mainly in inactivated states, leading to an allosterically caused increase in the inactivation probability, reflected in a negative shift of the steady-state inactivation curve, prolonged recovery from inactivation, and a frequency-dependent block. How local anesthetics bind to N-type inactivating K channels is less explored. In this study, we have compared bupivacaine effects on inactivating (Shaker and K_v3.4) and noninactivating (Shaker-IR and K_v3.2) channels, expressed in Xenopus oocytes. Bupivacaine was found to block these channels time-dependently without shifting the steady-state inactivation curve markedly, without a prolonged recovery from inactivation, and without a frequency-dependent block. An analysis, including computational testing of kinetic models, suggests binding to the channel mainly in the open state, with affinities close to those estimated for corresponding noninactivating channels (300 and 280 μM for Shaker and Shaker-IR, and 60 and 90 μM for K_v3.4 and K_v3.2). The similar magnitudes of K_d, as well as of blocking and unblocking rate constants for inactivating and noninactivating Shaker channels, most likely exclude allosteric interactions between the inactivation mechanism and the binding site. The relevance of these results for understanding the action of local anesthetics on Na channels is discussed.     

Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions

Inactivation is an intrinsic property of numerous voltage-gated K⁺ (Kv) channels and can occur by N-type or/and C-type mechanisms. N-type inactivation is a fast, voltage independent process, coupled to activation, with each inactivation particle of a tetrameric channel acting independently. In N-type inactivation, a single inactivation particle is necessary and sufficient to occlude the pore. C-type inactivation is a slower process, involving the outermost region of the pore and is mediated by a concerted, highly cooperative interaction between all four subunits. Inactivation of Kv7.1 channels does not exhibit the hallmarks of N- and C-type inactivation. Inactivation of WT Kv7.1 channels can be revealed by hooked tail currents that reflects the recovery from a fast and voltage-independent inactivation process. However, several Kv7.1 mutants such as the pore mutant L273F generate an additional voltage-dependent slow inactivation. The subunit interactions during this slow inactivation gating remain unexplored. The goal of the present study was to study the nature of subunit interactions along Kv7.1 inactivation gating, using concatenated tetrameric Kv7.1 channel and introducing sequentially into each of the four subunits the slow inactivating pore mutation L273F. Incorporating an incremental number of inactivating mutant subunits did not affect the inactivation kinetics but slowed down the recovery kinetics from inactivation. Results indicate that Kv7.1 inactivation gating is not compatible with a concerted cooperative process. Instead, adding an inactivating subunit L273F into the Kv7.1 tetramer incrementally stabilizes the inactivated state, which suggests that like for activation gating, Kv7.1 slow inactivation gating is not a concerted process.     

Nav1.3 and FGF14 are primary determinants of the TTX-sensitive sodium current in mouse adrenal chromaffin cells

Adrenal chromaffin cells (CCs) in rodents express rapidly inactivating, tetrodotoxin (TTX)-sensitive sodium channels. The resulting current has generally been attributed to Nav1.7, although a possible role for Nav1.3 has also been suggested. Nav channels in rat CCs rapidly inactivate via two independent pathways which differ in their time course of recovery. One subpopulation recovers with time constants similar to traditional fast inactivation and the other ∼10-fold slower, but both pathways can act within a single homogenous population of channels. Here, we use Nav1.3 KO mice to probe the properties and molecular components of Nav current in CCs. We find that the absence of Nav1.3 abolishes all Nav current in about half of CCs examined, while a small, fast inactivating Nav current is still observed in the rest. To probe possible molecular components underlying slow recovery from inactivation, we used mice null for fibroblast growth factor homology factor 14 (FGF14). In these cells, the slow component of recovery from fast inactivation is completely absent in most CCs, with no change in the time constant of fast recovery. The use dependence of Nav current reduction during trains of stimuli in WT cells is completely abolished in FGF14 KO mice, directly demonstrating a role for slow recovery from inactivation in determining Nav current availability. Our results indicate that FGF14-mediated inactivation is the major determinant defining use-dependent changes in Nav availability in CCs. These results establish that Nav1.3, like other Nav isoforms, can also partner with FGF subunits, strongly regulating Nav channel function.     

Modeling-independent elucidation of inactivation pathways in recombinant and native A-type Kv channels

A-type voltage-gated K⁺ (Kv) channels self-regulate their activity by inactivating directly from the open state (open-state inactivation [OSI]) or by inactivating before they open (closed-state inactivation [CSI]). To determine the inactivation pathways, it is often necessary to apply several pulse protocols, pore blockers, single-channel recording, and kinetic modeling. However, intrinsic hurdles may preclude the standardized application of these methods. Here, we implemented a simple method inspired by earlier studies of Na⁺ channels to analyze macroscopic inactivation and conclusively deduce the pathways of inactivation of recombinant and native A-type Kv channels. We investigated two distinct A-type Kv channels expressed heterologously (Kv3.4 and Kv4.2 with accessory subunits) and their native counterparts in dorsal root ganglion and cerebellar granule neurons. This approach applies two conventional pulse protocols to examine inactivation induced by (a) a simple step (single-pulse inactivation) and (b) a conditioning step (double-pulse inactivation). Consistent with OSI, the rate of Kv3.4 inactivation (i.e., the negative first derivative of double-pulse inactivation) precisely superimposes on the profile of the Kv3.4 current evoked by a single pulse because the channels must open to inactivate. In contrast, the rate of Kv4.2 inactivation is asynchronous, already changing at earlier times relative to the profile of the Kv4.2 current evoked by a single pulse. Thus, Kv4.2 inactivation occurs uncoupled from channel opening, indicating CSI. Furthermore, the inactivation time constant versus voltage relation of Kv3.4 decreases monotonically with depolarization and levels off, whereas that of Kv4.2 exhibits a J-shape profile. We also manipulated the inactivation phenotype by changing the subunit composition and show how CSI and CSI combined with OSI might affect spiking properties in a full computational model of the hippocampal CA1 neuron. This work unambiguously elucidates contrasting inactivation pathways in neuronal A-type Kv channels and demonstrates how distinct pathways might impact neurophysiological activity.     

Interaction between ion channel-inactivating peptides and anionic phospholipid vesicles as model targets.

Studies of rapid (N-type) inactivation induced by different synthetic inactivating peptides in several voltage-dependent cation channels have concluded that the channel inactivation "entrance" (or "receptor" site for the inactivating peptide) consists of a hydrophobic vestibule within the internal mouth of the channel, separated from the cytoplasm by a region with a negative surface potential. These protein domains are conformed from alternative sequences in the different channels and thus are relatively unrestricted in terms of primary structure. We are reporting here on the interaction between the inactivating peptide of the Shaker B K+ channel (ShB peptide) or the noninactivating ShB-L7E mutant with anionic phospholipid vesicles, a model target that, as the channel's inactivation "entrance," contains a hydrophobic domain (the vesicle bilayer) separated from the aqueous media by a negatively charged vesicle surface. When challenged by the anionic phospholipid vesicles, the inactivating ShB peptide 1) binds to the vesicle surface with a relatively high affinity, 2) readily adopts a strongly hydrogen-bonded beta-structure, likely an intramolecular beta "hairpin," and 3) becomes inserted into the hydrophobic bilayer by its folded N-terminal portion, leaving its positively charged C-terminal end exposed to the extravesicular aqueous medium. Similar experiments carried out with the noninactivating, L7E-ShB mutant peptide show that this peptide 1) binds also to the anionic vesicles, although with a lower affinity than does the ShB peptide, 2) adopts only occasionally the characteristic beta-structure, and 3) has completely lost the ability to traverse the anionic interphase at the vesicle surface and to insert into the hydrophobic vesicle bilayer. Because the negatively charged surface and the hydrophobic domains in the model target may partly imitate those conformed at the inactivation "entrance" of the channel proteins, we propose that channel inactivation likely includes molecular events similar to those observed in the interaction of the ShB peptide with the phospholipid vesicles, i.e., binding of the peptide to the region of negative surface potential, folding of the bound peptide as a beta-structure, and its insertion into the channel's hydrophobic vestibule. Likewise, we relate the lack of channel inactivation seen with the mutant ShB-L7E peptide to the lack of ability shown by this peptide to cross through the anionic interphase and insert into the hydrophobic domains of the model vesicle target.     

The cytosolic inactivation domains of BKi channels in rat chromaffin cells do not behave like simple, open-channel blockers.

Most BK-type voltage- and Ca(2+)-dependent K+ channels in rat chromaffin cells exhibit rapid inactivation. This inactivation is abolished by brief trypsin application to the cytosolic face of membrane patches. Here we examine the effects of cytosolic channel blockade and pore occupancy on this inactivation process, using inside-out patches and whole-cell recordings. Occupancy of a superficial pore-blocking site by cytosolic quaternary blockers does not slow inactivation. Occupancy of a deeper pore-blocking site by cytosolic application of Cs+ is also without effect on the onset of inactivation. Although the rate of inactivation is relatively unaffected by changes in extracellular K+, the rate of recovery from inactivation (at -80 and -140 mV with 10 microM Ca2+) is faster with increases in extracellular K+ but is unaffected by the impermeant ion, Na+. When tail currents are compared after repolarization, either while channels are open or after inactivation, no channel reopening is detectable during recovery from inactivation. BK inactivation appears to be mechanistically distinct from that of other inactivating voltage-dependent channels. Although involving a trypsin-sensitive cytosolic structure, the block to permeation does not appear to occur directly at the cytosolic mouth or inner half of the ion permeation pathway.     

Changes in the inactivation of rat Kv1.4 K(+) channels induced by varying the number of inactivation particles

N-type inactivation of rat Kv1.4 channels with one, two, or four inactivation balls was investigated using homogeneous populations of channels expressed in Xenopus oocytes. Tandem dimeric and tetrameric constructs of Kv1.4 were made. Channels encoded by tandem cDNAs Kv1. 4-Kv1.4Delta1-145 and Kv1.4-[Kv1.4Delta1-145](3) have two or only one tethered inactivation ball, respectively, whereas Kv1.4 itself encodes channels having four inactivation balls. The time constants for inactivation of macroscopic currents were increased significantly as the number of inactivation balls was decreased, whereas the time constants for recovery from inactivation were not modified. The ratios of the rate constants of inactivation (k(inact)) of Kv1.4-Kv1.4Delta1-145 and Kv1.4-[Kv1.4Delta1-145](3) channels to that of the Kv1.4 channel were 0.65 and 0.4, respectively, whereas the ratios of the rate constant of recovery (k(rec)) of these channels to that of Kv1.4 were almost unity. The rate constants k(inact) for channels having two and four inactivation balls are smaller than those that would be expected if inactivation balls on each channel are independent, suggesting some interaction occurs between inactivation balls. Furthermore, noninactivating current became apparent as the number of inactivation balls on a channel was decreased.     

Cultured Giant Fiber Lobe of Squid Expresses Three Distinct Potassium Channel Activities in Selective Combinations

Neurons from the giant fiber lobe (GFL) of squid Loligo bleekeri were dissociated and cultured. The ionic currents were recorded using whole-cell patch clamp methods. The sodium current and the noninactivating potassium current like those elicited by the giant axon were among the currents expressed in axonal bulbs and bulblike structures upon dissociation. Meanwhile axonless cell bodies did not elicit such currents. Axonless cell bodies and some bulblike structures elicited two kinds of inactivating potassium currents, the slow- and the fast-inactivating current, which differed in their inactivation kinetics and pharmacology. Within 24 hr of plating, the current composition remained the same. While the noninactivating current was not sensitive to 4-aminopyridine, the two inactivating currents were sensitive, the slow-inactivating current being more sensitive. Selective combinations of the sodium current and the three potassium currents expressed in different structures of the acutely dissociated GFL could have resulted from cellular control of synthesis and transportation of the channel proteins to the somatic and the axonal membrane. The sodium current and the noninactivating potassium current could be recorded from some axonless cell bodies maintained in culture for over three days, indicating that the separation of the giant axon from its somata could result in the transportation of the channels normally expressed on the giant axon membrane to the somatic membrane. Received: 24 October 1995/Revised: 5 March 1996     

A sodium channel defect in hyperkalemic periodic paralysis: potassium-induced failure of inactivation.

Hyperkalemic periodic analysis (HPP) is an autosomal dominant disorder characterized by episodic weakness lasting minutes to days in association with a mild elevation in serum K+. In vitro measurements of whole-cell currents in HPP muscle have demonstrated a persistent, tetrodotoxin-sensitive Na+ current, and we have recently shown by linkage analysis that the Na+ channel alpha subunit gene may contain the HPP mutation. In this study, we have made patch-clamp recordings from cultured HPP myotubes and found a defect in the normal voltage-dependent inactivation of Na+ channels. Moderate elevation of extracellular K+ favors an aberrant gating mode in a small fraction of the channels that is characterized by persistent reopenings and prolonged dwell times in the open state. The Na+ current, through noninactivating channels, may cause the skeletal muscle weakness in HPP by depolarizing the cell, thereby inactivating normal Na+ channels, which are then unable to generate an action potential. Thus the dominant expression of HPP is manifest by inactivation of the wild-type Na+ channel through the influence of the mutant gene product on membrane voltage.     

Ionic conductances of squid giant fiber lobe neurons

The cell bodies of the neurons in the giant fiber lobe (GFL) of the squid stellate ganglion give rise to axons that fuse and thereby form the third-order giant axon, whose initial portion functions as the postsynaptic element of the squid giant synapse. We have developed a preparation of dissociated, cultured cells from this lobe and have studied the voltage-dependent conductances using patch-clamp techniques. This system offers a unique opportunity for comparing the properties and regional differentiation of ionic channels in somatic and axonal membranes within the same cell. Some of these cells contain a small inward Na current which resembles that found in axon with respect to tetrodotoxin sensitivity, voltage dependence, and inactivation. More prominent is a macroscopic inward current, carried by Ca2+, which is likely to be the result of at least two kinetically distinct types of channels. These Ca channels differ in their closing kinetics, voltage range and time course of activation, and the extent to which their conductance inactivates. The dominant current in these GFL neurons is outward and is carried by K+. It can be accounted for by a single type of voltage-dependent channel. This conductance resembles the K conductance of the axon, except that it partially inactivates during relatively short depolarizations. Ensemble fluctuation analysis of K currents obtained from excised outside-out patches is consistent with a single type of K channel and yields estimates for the single channel conductance of approximately 13 pS, independently of membrane potential. A preliminary analysis of single channel data supports the conclusion that there is a single type of voltage-dependent, inactivating K channel in the GFL neurons.     

Inactivation in ShakerB K+ channels: a test for the number of inactivating particles on each channel.

Fast inactivation in ShakerB K channels results from pore-block caused by "ball peptides" attached to the inner part of each K channel. We have examined the question of how many functional inactivating balls are on each channel and how this number affects inactivation and recovery from inactivation. To that purpose we expressed ShakerB in the insect cell line Sf9 and gradually removed inactivation by perfusing the cell interior with the hydrolytic enzyme papain under whole cell patch clamp. Inactivation slows down as the balls are removed by an amount consistent with the presence of four balls on each channel. Recovery from inactivation has the same time course early and late in papain action; it does not depend on the number of balls remaining on the channel, consistent with the idea that reinactivation is not significant during recovery from inactivation. Our conclusion is that ShakerB has four ball peptides, each capable of causing inactivation. Statistically, the balls are identical and independent. The stability of N-type inactivation by the remaining balls is not appreciably affected by removing some of the balls from a channel.