Phosphatidylinositol 4,5-Bisphosphate Activates Slo3 Currents and Its Hydrolysis Underlies the Epidermal Growth Factor-induced Current Inhibition

The Slo3 gene encodes a high conductance potassium channel, which is activated by both voltage and intracellular alkalinization. Slo3 is specifically expressed in mammalian sperm cells, where it gives rise to pH-dependent outwardly rectifying K⁺ currents. Sperm Slo3 is the main current responsible for the capacitation-induced hyperpolarization, which is required for the ensuing acrosome reaction, an exocytotic process essential for fertilization. Here we show that in intact spermatozoa and in a heterologous expression system, the activation of Slo3 currents is regulated by phosphatidylinositol 4,5-bisphosphate (PIP₂). Depletion of endogenous PIP₂ in inside-out macropatches from Xenopus oocytes inhibited heterologously expressed Slo3 currents. Whole-cell recordings of sperm Slo3 currents or of Slo3 channels co-expressed in Xenopus oocytes with epidermal growth factor receptor, demonstrated that stimulation by epidermal growth factor (EGF) could inhibit channel activity in a PIP₂-dependent manner. High concentrations of PIP₂ in the patch pipette not only resulted in a strong increase in sperm Slo3 current density but also prevented the EGF-induced inhibition of this current. Mutation of positively charged residues involved in channel-PIP₂ interactions enhanced the EGF-induced inhibition of Slo3 currents. Overall, our results suggest that PIP₂ is an important regulator for Slo3 activation and that receptor-mediated hydrolysis of PIP₂ leads to inhibition of Slo3 currents both in native and heterologous expression systems.     

Identification of the Intracellular Na+ Sensor in Slo2.1 Potassium Channels

Slo2 potassium channels have a very low open probability under normal physiological conditions, but are readily activated in response to an elevated [Na⁺]_i (e.g. during ischemia). An intracellular Na⁺ coordination motif (DX(R/K)XXH) was previously identified in Kir3.2, Kir3.4, Kir5.1, and Slo2.2 channel subunits. Based loosely on this sequence, we identified five potential Na⁺ coordination motifs in the C terminus of the Slo2.1 subunit. The Asp residue in each sequence was substituted with Arg, and single mutant channels were heterologously expressed in Xenopus oocytes. The Na⁺ sensitivity of each of the mutant channels was assessed by voltage clamp of oocytes using micropipettes filled with 2 m NaCl. Wild-type channels and four of the mutant Slo2.1 channels were rapidly activated by leakage of NaCl solution into the cytoplasm. D757R Slo2.1 channels were not activated by NaCl, but were activated by the fenamate niflumic acid, confirming their functional expression. In whole cell voltage clamp recordings of HEK293 cells, wild-type but not D757R Slo2.1 channels were activated by a [NaCl]_i of 70 mm. Thus, a single Asp residue can account for the sensitivity of Slo2.1 channels to intracellular Na⁺. In excised inside-out macropatches of HEK293 cells, activation of wild-type Slo2.1 currents by 3 mm niflumic acid was 14-fold greater than activation achieved by increasing [NaCl]_i from 3 to 100 mm. Thus, relative to fenamates, intracellular Na⁺ is a poor activator of Slo2.1.     

Activation of Slo2.1 channels by niflumic acid

Slo2.1 channels conduct an outwardly rectifying K⁺ current when activated by high [Na⁺]_i. Here, we show that gating of these channels can also be activated by fenamates such as niflumic acid (NFA), even in the absence of intracellular Na⁺. In Xenopus oocytes injected with <10 ng cRNA, heterologously expressed human Slo2.1 current was negligible, but rapidly activated by extracellular application of NFA (EC₅₀ = 2.1 mM) or flufenamic acid (EC₅₀ = 1.4 mM). Slo2.1 channels activated by 1 mM NFA exhibited weak voltage dependence. In high [K⁺]_e, the conductance–voltage (G-V) relationship had a V_1/2 of +95 mV and an effective valence, z, of 0.48 e. Higher concentrations of NFA shifted V_1/2 to more negative potentials (EC₅₀ = 2.1 mM) and increased the minimum value of G/G_max (EC₅₀ = 2.4 mM); at 6 mM NFA, Slo2.1 channel activation was voltage independent. In contrast, V_1/2 of the G-V relationship was shifted to more positive potentials when [K⁺]_e was elevated from 1 to 300 mM (EC₅₀ = 21.2 mM). The slope conductance measured at the reversal potential exhibited the same [K⁺]_e dependency (EC₅₀ = 23.5 mM). Conductance was also [Na⁺]_e dependent. Outward currents were reduced when Na⁺ was replaced with choline or mannitol, but unaffected by substitution with Rb⁺ or Li⁺. Neutralization of charged residues in the S1–S4 domains did not appreciably alter the voltage dependence of Slo2.1 activation. Thus, the weak voltage dependence of Slo2.1 channel activation is independent of charged residues in the S1–S4 segments. In contrast, mutation of R190 located in the adjacent S4–S5 linker to a neutral (Ala or Gln) or acidic (Glu) residue induced constitutive channel activity that was reduced by high [K⁺]_e. Collectively, these findings indicate that Slo2.1 channel gating is modulated by [K⁺]_e and [Na⁺]_e, and that NFA uncouples channel activation from its modulation by transmembrane voltage and intracellular Na⁺.     

SLO-2 isoforms with unique Ca2+- and voltage-dependence characteristics confer sensitivity to hypoxia in C. elegans

Slo channels are large conductance K⁺ channels that display marked differences in their gating by intracellular ions. Among them, the Slo1 and C. elegans SLO-2 channels are gated by calcium (Ca²⁺), while mammalian Slo2 channels are activated by both sodium (Na⁺) and chloride (Cl⁻). Here, we report that SLO-2 channels, SLO-2a and a novel N-terminal variant isoform, SLO-2b, are activated by Ca²⁺ and voltage, but in contrast to previous reports they do not exhibit Cl⁻ sensitivity. Most importantly, SLO-2 provides a unique case in the Slo family for sensing Ca²⁺ with the high-affinity Ca²⁺ regulatory site in the RCK1 but not the RCK2 domain, formed through interactions with residues E319 and E487 (that correspond to D362 and E535 of Slo1, respectively). The SLO-2 RCK2 domain lacks the Ca²⁺ bowl structure and shows minimal Ca²⁺ dependence. In addition, in contrast to SLO-1, SLO-2 loss-of-function mutants confer resistance to hypoxia in C. elegans. Thus, the C. elegans SLO-2 channels possess unique biophysical and functional properties.     

SLO3 K+ Channels Control Calcium Entry through CATSPER Channels in Sperm

Here we show how a sperm-specific potassium channel (SLO3) controls Ca²⁺ entry into sperm through a sperm-specific Ca²⁺ channel, CATSPER, in a totally unanticipated manner. The genetic deletion of either of those channels confers male infertility in mice. During sperm capacitation SLO3 hyperpolarizes the sperm, whereas CATSPER allows Ca²⁺ entry. These two channels may be functionally connected, but it had not been demonstrated that SLO3-dependent hyperpolarization is required for Ca²⁺ entry through CATSPER channels, nor has a functional mechanism linking the two channels been shown. In this study we show that Ca²⁺ entry through CATSPER channels is deficient in Slo3 mutant sperm lacking hyperpolarization; we also present evidence supporting the hypothesis that SLO3 channels activate CATSPER channels indirectly by promoting a rise in intracellular pH through a voltage-dependent mechanism. This mechanism may work through a Na⁺/H⁺ exchanger (sNHE) and/or a bicarbonate transporter, which utilizes the inward driving force of the Na⁺ gradient, rendering it intrinsically voltage-dependent. In addition, the sperm-specific Na⁺/H⁺ exchanger (sNHE) possess a putative voltage sensor that might be activated by membrane hyperpolarization, thus increasing the voltage sensitivity of internal alkalization.     

Sensitivity of cloned muscle,heart and neuronal voltage-gated sodium channels to block by polyamines

Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K⁺ current, a phenomenon known as inward rectification characteristic of a major class of K_IR K⁺ channels. We previously described block of heterologously expressed voltage-gated Na⁺ channels (Na_V) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal Na_V channels. In this study, we compared the sensitivity of four different cloned mammalian Na_V isoforms to PAs to investigate whether PA block is a common feature of Na_V channel pharmacology. We find that outward Na⁺ current of muscle (Na_V1.4), heart (Na_V1.5), and neuronal (Na_V1.2, Na_V1.7) Na_V isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac Na_V1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the Na_V1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term Na_V channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.     

Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa

During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca²⁺. The elevation of Ca²⁺ is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K⁺ current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca²⁺ current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1–4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K⁺ current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K⁺ current present in the Slo3 ^−/− sperm arise from CATSPER? Second, can any additional membrane K⁺ currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 ^−/− sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca²⁺ influx in response to alkalization.     

Sensitivity of cloned muscle,heart and neuronal voltage-gated sodium channels to block by polyamines: A possible basis for modulation of excitability in vivo

Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K⁺ current, a phenomenon known as inward rectification characteristic of a major class of K_IR K⁺ channels. We previously described block of heterologously expressed voltage-gated Na⁺ channels (Na_V) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal Na_V channels. In this study, we compared the sensitivity of four different cloned mammalian Na_V isoforms to PAs to investigate whether PA block is a common feature of Na_V channel pharmacology. We find that outward Na⁺ current of muscle (Na_V1.4), heart (Na_V1.5), and neuronal (Na_V1.2, Na_V1.7) Na_V isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac Na_V1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the Na_V1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term Na_V channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.     

Effect of Alkali Metal Cations on Slow Inactivation of Cardiac Na+ Channels

Human heart Na⁺ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na⁺ currents measured using 150 mM intracellular Na⁺. The kinetics of decaying outward Na⁺ current in response to 1-s depolarizations in the F1485Q mutant depends on the predominant cation in the extracellular solution, suggesting an effect on slow inactivation. The decay rate is lower for the alkali metal cations Li⁺, Na⁺, K⁺, Rb⁺, and Cs⁺ than for the organic cations Tris, tetramethylammonium, N-methylglucamine, and choline. In whole cell recordings, raising [Na⁺]_o from 10 to 150 mM increases the rate of recovery from slow inactivation at −140 mV, decreases the rate of slow inactivation at relatively depolarized voltages, and shifts steady-state slow inactivation in a depolarized direction. Single channel recordings of F1485Q show a decrease in the number of blank (i.e., null) records when [Na⁺]_o is increased. Significant clustering of blank records when depolarizing at a frequency of 0.5 Hz suggests that periods of inactivity represent the sojourn of a channel in a slow-inactivated state. Examination of the single channel kinetics at +60 mV during 90-ms depolarizations shows that neither open time, closed time, nor first latency is significantly affected by [Na⁺]_o. However raising [Na⁺]_o decreases the duration of the last closed interval terminated by the end of the depolarization, leading to an increased number of openings at the depolarized voltage. Analysis of single channel data indicates that at a depolarized voltage a single rate constant for entry into a slow-inactivated state is reduced in high [Na⁺]_o, suggesting that the binding of an alkali metal cation, perhaps in the ion-conducting pore, inhibits the closing of the slow inactivation gate.     

Computer simulations of neuron-glia interactions mediated by ion flux

Extracellular potassium concentration, [K⁺]_o, and intracellular calcium, [Ca²⁺]_i, rise during neuron excitation, seizures and spreading depression. Astrocytes probably restrain the rise of K⁺ in a way that is only partly understood. To examine the effect of glial K⁺ uptake, we used a model neuron equipped with Na⁺, K⁺, Ca²⁺ and Cl⁻ conductances, ion pumps and ion exchangers, surrounded by interstitial space and glia. The glial membrane was either “passive”, incorporating only leak channels and an ion exchange pump, or it had rectifying K⁺ channels. We computed ion fluxes, concentration changes and osmotic volume changes. Increase of [K⁺]_o stimulated the glial uptake by the glial 3Na/2K ion pump. The [K⁺]_o flux through glial leak and rectifier channels was outward as long as the driving potential was outwardly directed, but it turned inward when rising [K⁺]_o/[K⁺]_i ratio reversed the driving potential. Adjustments of glial membrane parameters influenced the neuronal firing patterns, the length of paroxysmal afterdischarge and the ignition point of spreading depression. We conclude that voltage gated K⁺ currents can boost the effectiveness of the glial “potassium buffer” and that this buffer function is important even at moderate or low levels of excitation, but especially so in pathological states.     

Constitutive inactivation of the hKv1.5 mutant channel, H463G, in K+-free solutions at physiological pH

Extracellular acidification and reduction of extracellular K⁺ are known to decrease the currents of some voltage-gated potassium channels. Although the macroscopic conductance of WT hKv1.5 channels is not very sensitive to [K⁺]_o at pH 7.4, it is very sensitive to [K⁺]_o at pH 6.4, and in the mutant, H463G, the removal of K⁺ _o virtually eliminates the current at pH 7.4. We investigated the mechanism of current regulation by K⁺ _o in the Kv1.5 H463G mutant channel at pH 7.4 and the wild-type channel at pH 6.4 by taking advantage of Na⁺ permeation through inactivated channels. Although the H463G currents were abolished in zero [K⁺]_o, robust Na⁺ tail currents through inactivated channels were observed. The appearnnce of H463G Na⁺ currents with a slow rising phase on repolarization after a very brief depolarization (2 ms) suggests that channels could activate directly from closed-inactivated states. In wild-type channels, when intracellular K⁺ was replaced by NMG⁺ and the inward Na⁺ current was recorded, addition of 1 mM K⁺ prevented inactivation, but changing pH from 7.4 to 6.4 reversed this action. The data support the idea that C-type inactivation mediated at R487 in Kv1.5 channels is influenced by H463 in the outer pore. We conclude that both acidification and reduction of [K⁺]_o inhibit Kv1.5 channels through a common mechananism (i.e., by increasing channel inactivation, which occurs in the resting state or develops very rapidly after activation).     

Cell Cycle–related Changes in the Conducting Properties of r-eag K+ Channels

Release from arrest in G2 phase of the cell cycle causes profound changes in rat ether-à-go-go (r-eag) K⁺ channels heterologously expressed in Xenopus oocytes. The most evident consequence of the onset of maturation is the appearance of rectification in the r-eag current. The trigger for these changes is located downstream of the activation of mitosis-promoting factor (MPF). We demonstrate here that the rectification is due to a voltage-dependent block by intracellular Na⁺ ions. Manipulation of the intracellular Na⁺ concentration indicates that the site of Na⁺ block is located ∼45% into the electrical distance of the pore and is only present in oocytes undergoing maturation. Since the currents through excised patches from immature oocytes exhibited a fast rundown, we studied CHO-K1 cells permanently transfected with r-eag. These cells displayed currents with a variable degree of block by Na⁺ and variable permeability to Cs⁺. Partial synchronization of the cultures in G0/G1 or M phases of the cell cycle greatly reduced the variability. The combined data obtained from mammalian cells and oocytes strongly suggest that the permeability properties of r-eag K⁺ channels are modulated during cell cycle–related processes.     

Seizure-like afterdischarges simulated in a model neuron

To explore non-synaptic mechanisms in paroxysmal discharges, we used a computer model of a simplified hippocampal pyramidal cell, surrounded by interstitial space and a “glial-endothelial” buffer system. Ion channels for Na⁺, K⁺, Ca²⁺ and Cl⁻ _, ion antiport 3Na/Ca, and “active” ion pumps were represented in the neuron membrane. The glia had “leak” conductances and an ion pump. Fluxes, concentration changes and cell swelling were computed. The neuron was stimulated by injecting current. Afterdischarge (AD) followed stimulation if depolarization due to rising interstitial K⁺ concentration ([K⁺]_o) activated persistent Na⁺ current (I _Na,P). AD was either simple or self-regenerating; either regular (tonic) or burst-type (clonic); and always self-limiting. Self-regenerating AD required sufficient I _Na,P to ensure re-excitation. Burst firing depended on activation of dendritic Ca²⁺ currents and Ca-dependent K⁺ current. Varying glial buffer function influenced [K⁺]_o accumulation and afterdischarge duration. Variations in Na⁺ and K⁺ currents influenced the threshold and the duration of AD. The data show that high [K⁺]_o and intrinsic membrane currents can produce the feedback of self-regenerating afterdischarges without synaptic input. The simulated discharge resembles neuron behavior during paroxysmal firing in living brain tissue. Action Editor: David Terman     

The Mechanism of Na+/K+ Selectivity in Mammalian Voltage-Gated Sodium Channels Based on Molecular Dynamics Simulation

Voltage-gated sodium (Na_v) channels and their Na⁺/K⁺ selectivity are of great importance in the mammalian neuronal signaling. According to mutational analysis, the Na⁺/K⁺ selectivity in mammalian Na_v channels is mainly determined by the Lys and Asp/Glu residues located at the constriction site within the selectivity filter. Despite successful molecular dynamics simulations conducted on the prokaryotic Na_v channels, the lack of Lys at the constriction site of prokaryotic Na_v channels limits how much can be learned about the Na⁺/K⁺ selectivity in mammalian Na_v channels. In this work, we modeled the mammalian Na_v channel by mutating the key residues at the constriction site in a prokaryotic Na_v channel (Na_vRh) to its mammalian counterpart. By simulating the mutant structure, we found that the Na⁺ preference in mammalian Na_v channels is collaboratively achieved by the deselection from Lys and the selection from Asp/Glu within the constriction site.     

A comparative analysis of models of Na+ channel gating for mammalian and invertebrate nonmyelinated axons: Relationship to energy efficient action potentials

The rapidly activating, voltage gated Na⁺ current, I_Na, has recently been measured in mammalian nonmyelinated axons. Those results have been incorporated in simulations of the action potential, results that demonstrate a significant separation in time during the spike between I_Na and the repolarizing K⁺ current, I_K. The original Hodgkin and Huxley (1952) model of Na⁺ channel gating, m³h, where m and h are channel activation and inactivation, respectively, has been used in this analysis. This model was originally developed for invertebrate nonmyelinated axons, squid giant axons in particular. The model has not survived challenges based on results from invertebrate preparations using a double-step voltage clamp protocol and measurements of gating currents, results that demonstrate a kinetic link between activation and inactivation leading to a delayed onset of inactivation following a voltage step. These processes are independent of each other in the Hodgkin and Huxley (1952) model. Application of the double-step protocol to the m³h model for mammalian I_Na results reveals a surprising prediction, an apparent delay in onset of inactivation even though activation and inactivation are uncoupled in the model. Other results, most notably gating currents, will be required to demonstrate such a link, if indeed it exists for mammalian Na⁺ channels. The information obtained will be significant in determining the way in which the Na⁺ channel is sequestered away from its open state during repolarization, thereby allowing for a separation in time between I_Na and I_K during a spike, an energetically efficient mechanism of neuronal signaling in the mammalian brain.     

Anomalous Effect of Permeant Ion Concentration on Peak Open Probability of Cardiac Na+ Channels

Human heart Na⁺ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na⁺ currents measured using 150 mM intracellular Na⁺. Decreasing extracellular permeant ion concentration decreases outward Na⁺ current at positive voltages while increasing the driving force for the current. This anomalous effect of permeant ion concentration, especially obvious in a mutant (F1485Q) in which fast inactivation is partially abolished, is due to an alteration of open probability. The effect is only observed when a highly permeant cation (Na⁺, Li⁺, or hydrazinium) is substituted for a relatively impermeant cation (K⁺, Rb⁺, Cs⁺, N -methylglucamine, Tris, choline, or tetramethylammonium). With high concentrations of extracellular permeant cations, the peak open probability of Na⁺ channels increases with depolarization and then saturates at positive voltages. By contrast, with low concentrations of permeant ions, the open probability reaches a maximum at approximately 0 mV and then decreases with further depolarization. There is little effect of permeant ion concentration on activation kinetics at depolarized voltages. Furthermore, the lowered open probability caused by a brief depolarization to +60 mV recovers within 5 ms upon repolarization to −140 mV, indicative of a gating process with rapid kinetics. Tail currents at reduced temperatures reveal the rapid onset of this gating process during a large depolarization. A large depolarization may drive a permeant cation out of a site within the extracellular mouth of the pore, reducing the efficiency with which the channel opens.     

K Channels Are Responsible for an Inwardly Rectifying Current in the Plasma Membrane of Mesophyll Protoplasts of Avena sativa

In whole-cell recording, the conductance of the plasma membrane of protoplasts isolated from mesophyll cells of leaves of oat (Avena sativa) was greater for inward than outward current. The inward current in both the whole-cell mode and with isolated patches was dependent on [K⁺]_o. When the membrane voltage was more positive than −50 millivolts, the membrane conductance in the whole-cell mode was low, and K⁺ channels in cell-attached or outside-out patches had a low probability of being open. At a membrane voltage more negative than −50 millivolts, the membrane conductance increased by sevenfold in the whole-cell mode, and the probability of the channels being open increased. The inward current was highly selective for K⁺ compared with Cs⁺, Na⁺, choline or Cl⁻. Low concentrations of [Cs⁺]_o or [Na⁺]_o blocked the inward current in a strongly voltage-dependent fashion. Comparison of single-channel with the macroscopic current yields an estimate of about 200 inwardly rectifying K⁺ channels per cell at a density of 0.035 per square micrometer. At physiological membrane voltages and [K⁺]_o about 10 millimolar, the influx through these channels is sufficient to increase the internal [K⁺] by 2 millimolar per minute. These K⁺ channels are activated by membrane voltages in the normal physiological range and could contribute to K⁺ uptake whenever the membrane is more negative than the K⁺ equilibrium potential.     

Potassium Channels in Myelinated Nerve : Selective permeability to small cations

The permeability of K channels to various cations is studied in myelinated nerve. Ionic currents under voltage clamp are measured in Ringer solution containing tetrodotoxin and a high concentration of the test ion. Reversal potentials for current in K channels are determined and used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The ratios P_Tl:P_K:P_Rb:P_NHNH4 are 2.3:1.00:0.92:0.13. No other ions are found to be measurably permeant including Li⁺, Na⁺, Cs⁺, methylamine, guanidine, hydrazine, or hydroxylamine. The ratio P_Na/P_K is less than 0.01. Potassium conductance is depressed at pH values below 5.0. Leakage conductance is higher in K, Rb, Cs, NH₄, and Tl Ringer than in Na Ringer, but the selectivity sequence probably is not the same as for K channels. The hypothesis is offered that the narrowest part of the K channel is a circle of oxygen atoms about 3 Å in diameter with low electrostatic field strength.     

cAMP Control of HCN2 Channel Mg2+ Block Reveals Loose Coupling between the Cyclic Nucleotide-Gating Ring and the Pore

Hyperpolarization-activated cyclic nucleotide-regulated HCN channels underlie the Na⁺-K⁺ permeable I_H pacemaker current. As with other voltage-gated members of the 6-transmembrane K_V channel superfamily, opening of HCN channels involves dilation of a helical bundle formed by the intracellular ends of S6 albeit this is promoted by inward, not outward, displacement of S4. Direct agonist binding to a ring of cyclic nucleotide-binding sites, one of which lies immediately distal to each S6 helix, imparts cAMP sensitivity to HCN channel opening. At depolarized potentials, HCN channels are further modulated by intracellular Mg²⁺ which blocks the open channel pore and blunts the inhibitory effect of outward K⁺ flux. Here, we show that cAMP binding to the gating ring enhances not only channel opening but also the kinetics of Mg²⁺ block. A combination of experimental and simulation studies demonstrates that agonist acceleration of block is mediated via acceleration of the blocking reaction itself rather than as a secondary consequence of the cAMP enhancement of channel opening. These results suggest that the activation status of the gating ring and the open state of the pore are not coupled in an obligate manner (as required by the often invoked Monod-Wyman-Changeux allosteric model) but couple more loosely (as envisioned in a modular model of protein activation). Importantly, the emergence of second messenger sensitivity of open channel rectification suggests that loose coupling may have an unexpected consequence: it may endow these erstwhile “slow” channels with an ability to exert voltage and ligand-modulated control over cellular excitability on the fastest of physiologically relevant time scales.