Charged tetracaine as an inactivation enhancer in batrachotoxin-modified Na+ channels.

Two distinct types of local anesthetics (LAs) have previously been found to block batrachotoxin (BTX)-modified Na+ channels: type 1 LAs such as cocaine and bupivacaine interact preferentially with open channels, whereas type 2 LAs, such as benzocaine and tricaine, with inactivated channels. Herein, we describe our studies of a third type of LA, represented by tetracaine as a dual blocker that binds strongly with closed channels but also binds to a lesser extent with open channels when the membrane is depolarized. Enhanced inactivation of BTX-modified Na+ channels by tetracaine was determined by steady-state inactivation measurement and by the dose-response curve. The 50% inhibitory concentration (IC50) was estimated to be 5.2 microM at -70 mV, where steady-state inactivation was maximal, with a Hill coefficient of 0.98 suggesting that one tetracaine molecule binds with one inactivated channel. Tetracaine also interacted efficiently with Na+ channels when the membrane was depolarized; the IC50 was estimated to be 39.5 microM at +50 mV with a Hill coefficient of 0.94. Unexpectedly, charged tetracaine was found to be the primary active form in the blocking of inactivated channels. In addition, external Na+ ions appeared to antagonize the tetracaine block of inactivated channels. Consistent with these results, N-butyl tetracaine quaternary ammonium, a permanently charged tetracaine derivative, remained a strong inactivation enhancer. Another derivative of tetracaine, 2-(di-methylamino) ethyl benzoate, which lacked a 4-butylamino functional group on the phenyl ring, elicited block that was approximately 100-fold weaker than that of tetracaine. We surmise that 1) the binding site for inactivation enhancers is within the Na+ permeation pathway, 2) external Na+ ions antagonize the block of inactivation enhancers by electrostatic repulsion, 3) the 4-butylamino functional group on the phenyl ring is critical for block and for the enhancement of inactivation, and 4) there are probably overlapping binding sites for both inactivation enhancers and open-channel blockers within the Na+ pore.     

Plants do it differently. A new basis for potassium/sodium selectivity in the pore of an ion channel

Understanding of the molecular architecture necessary for selective K(+) permeation through the pore of ion channels is based primarily on analysis of the crystal structure of the bacterial K(+) channel KcsA, and structure:function studies of cloned animal K(+) channels. Little is known about the conduction properties of a large family of plant proteins with structural similarities to cloned animal cyclic nucleotide-gated channels (CNGCs). Animal CNGCs are nonselective cation channels that do not discriminate between Na(+) and K(+) permeation. These channels all have the same triplet of amino acids in the channel pore ion selectivity filter, and this sequence is different from that of the selectivity filter found in K(+)-selective channels. Plant CNGCs have unique pore selectivity filters; unlike those found in any other family of channels. At present, the significance of the unique pore selectivity filters of plant CNGCs, with regard to discrimination between Na(+) and K(+) permeation is unresolved. Here, we present an electrophysiological analysis of several members of this protein family; identifying the first cloned plant channel (AtCNGC1) that conducts Na(+). Another member of this ion channel family (AtCNGC2) is shown to have a selectivity filter that provides a heretofore unknown molecular basis for discrimination between K(+) and Na(+) permeation. Specific amino acids within the AtCNGC2 pore selectivity filter (Asn-416, Asp-417) are demonstrated to facilitate K(+) over Na(+) conductance. The selectivity filter of AtCNGC2 represents an alternative mechanism to the well-known GYG amino acid triplet of K(+) channels that has been identified as the critical basis for K(+) over Na(+) permeation through the pore of ion channels.     

Convergent evolution of sodium ion selectivity in metazoan neuronal signaling

Ion selectivity of metazoan voltage-gated Na(+) channels is critical for neuronal signaling and has long been attributed to a ring of four conserved amino acids that constitute the ion selectivity filter (SF) at the channel pore. Yet, in addition to channels with a preference for Ca(2+) ions, the expression and characterization of Na(+) channel homologs from the sea anemone Nematostella vectensis, a member of the early-branching metazoan phylum Cnidaria, revealed a sodium-selective channel bearing a noncanonical SF. Mutagenesis and physiological assays suggest that pore elements additional to the SF determine the preference for Na(+) in this channel. Phylogenetic analysis assigns the Nematostella Na(+)-selective channel to a channel group unique to Cnidaria, which diverged >540 million years ago from Ca(2+)-conducting Na(+) channel homologs. The identification of Cnidarian Na(+)-selective ion channels distinct from the channels of bilaterian animals indicates that selectivity for Na(+) in neuronal signaling emerged independently in these two animal lineages.     

State-dependent block of CNG channels by dequalinium

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels with a high permeability for Ca(2+). Not surprisingly, they are blocked by a number of Ca(2+) channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance Ca(2+)-activated K(+) channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV. Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3-5 times higher than that for open channels. Mutations in the S4-S5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10-20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway.     

Potassium-selective block of barium permeation through single KcsA channels

Ba(2+), a doubly charged analogue of K(+), specifically blocks K(+) channels by virtue of electrostatic stabilization in the permeation pathway. Ba(2+) block is used here as a tool to determine the equilibrium binding affinity for various monovalent cations at specific sites in the selectivity filter of a noninactivating mutant of KcsA. At high concentrations of external K(+), the block-time distribution is double exponential, marking at least two Ba(2+) sites in the selectivity filter, in accord with a Ba(2+)-containing crystal structure of KcsA. By analyzing block as a function of extracellular K(+), we determined the equilibrium dissociation constant of K(+) and of other monovalent cations at an extracellular site, presumably S1, to arrive at a selectivity sequence for binding at this site: Rb(+) (3 μM) > Cs(+) (23 μM) > K(+) (29 μM) > NH(4)(+) (440 μM) > Na(+) and Li(+) (>1 M). This represents an unusually high selectivity for K(+) over Na(+), with |ΔΔG(0)| of at least 7 kcal mol(-1). These results fit well with other kinetic measurements of selectivity as well as with the many crystal structures of KcsA in various ionic conditions.     

Structural implications of weak Ca2+ block in Drosophila cyclic nucleotide–gated channels

Calcium permeability and the concomitant calcium block of monovalent ion current (“Ca²⁺ block”) are properties of cyclic nucleotide–gated (CNG) channel fundamental to visual and olfactory signal transduction. Although most CNG channels bear a conserved glutamate residue crucial for Ca²⁺ block, the degree of block displayed by different CNG channels varies greatly. For instance, the Drosophila melanogaster CNG channel shows only weak Ca²⁺ block despite the presence of this glutamate. We previously constructed a series of chimeric channels in which we replaced the selectivity filter of the bacterial nonselective cation channel NaK with a set of CNG channel filter sequences and determined that the resulting NaK2CNG chimeras displayed the ion selectivity and Ca²⁺ block properties of the parent CNG channels. Here, we used the same strategy to determine the structural basis of the weak Ca²⁺ block observed in the Drosophila CNG channel. The selectivity filter of the Drosophila CNG channel is similar to that of most other CNG channels except that it has a threonine at residue 318 instead of a proline. We constructed a NaK chimera, which we called NaK2CNG-Dm, which contained the Drosophila selectivity filter sequence. The high resolution structure of NaK2CNG-Dm revealed a filter structure different from those of NaK and all other previously investigated NaK2CNG chimeric channels. Consistent with this structural difference, functional studies of the NaK2CNG-Dm chimeric channel demonstrated a loss of Ca²⁺ block compared with other NaK2CNG chimeras. Moreover, mutating the corresponding threonine (T318) to proline in Drosophila CNG channels increased Ca²⁺ block by 16 times. These results imply that a simple replacement of a threonine for a proline in Drosophila CNG channels has likely given rise to a distinct selectivity filter conformation that results in weak Ca²⁺ block.     

Anomalous mole-fraction effects in recombinant and native cyclic nucleotide-gated channels in rat olfactory receptor neurons.

Anomalous mole-fraction effects (AMFE) were studied, using the inside-out configuration of the patchclamp technique, in both recombinant wild-type alpha-homomeric rat olfactory adenosine 3',5'-cyclic monophosphate (cAMP)-gated channels (rOCNC1) expressed in human embryonic kidney cells (HEK 293) and native cyclic nucleotide-gated (CNG) channels in acutely isolated rat olfactory receptor neurons. Single-channel and macroscopic currents were activated by 200 microM and 500 microM cAMP, respectively. Macroscopic currents, measured with mixtures of Na(+)-NH(4)(+) or Cs(+)-Li(+) in the cytoplasmic bathing solution, displayed AMFE in the rOCNC1 channels at both positive and negative membrane potentials. The rOCNC1 single-channel conductance showed a distinct minimum (or maximum) in an 80% Na(+)-20% NH(4)(+) mixture (or a 60% Cs(+)-40% Li(+) mixture), but only at positive membrane potentials. Macroscopic measurements in native olfactory CNG channels with mixtures of Na(+)-NH(4)(+) indicated similar AMFE. These results suggest that both native CNG channels and recombinant alpha-homomeric channels allow several ions to be present simultaneously within the channel pore. They also further validate the dominant role of the alpha-subunit in permeation through these channels, provide the first evidence to suggest that rOCNC1 channels have multi-ion properties and further justify the use of the rOCNC1 channel as an effective model for structure-function studies of ion permeation and selectivity in olfactory CNG channels.     

Effect of Na(+) flow on Cd(2+) block of tetrodotoxin-resistant Na(+) channels

Tetrodotoxin-resistant (TTX-R) Na(+) channels are 1,000-fold less sensitive to TTX than TTX-sensitive (TTX-S) Na(+) channels. On the other hand, TTX-R channels are much more susceptible to external Cd(2+) block than TTX-S channels. A cysteine (or serine) residue situated just next to the aspartate residue of the presumable selectivity filter "DEKA" ring of the TTX-R channel has been identified as the key ligand determining the binding affinity of both TTX and Cd(2+). In this study we demonstrate that the binding affinity of Cd(2+) to the TTX-R channels in neurons from dorsal root ganglia has little intrinsic voltage dependence, but is significantly influenced by the direction of Na(+) current flow. In the presence of inward Na(+) current, the apparent dissociation constant of Cd(2+) ( approximately 200 microM) is approximately 9 times smaller than that in the presence of outward Na(+) current. The Na(+) flow-dependent binding affinity change of Cd(2+) block is true no matter whether the direction of Na(+) current is secured by asymmetrical chemical gradient (e.g., 150 mM Na(+) vs. 150 mM Cs(+) on different sides of the membrane, 0 mV) or by asymmetrical electrical gradient (e.g., 150 mM Na(+) on both sides of the membrane, -20 mV vs. 20 mV). These findings suggest that Cd(2+) is a pore blocker of TTX-R channels with its binding site located in a multiion, single-file region near the external pore mouth. Quantitative analysis of the flow dependence with the flux-coupling equation reveals that at least two Na(+) ions coexist with the blocking Cd(2+) ion in this pore region in the presence of 150 mM ambient Na(+). Thus, the selectivity filter of the TTX-R Na(+) channels in dorsal root ganglion neurons might be located in or close to a multiion single-file pore segment connected externally to a wide vestibule, a molecular feature probably shared by other voltage-gated cationic channels, such as some Ca(2+) and K(+) channels.     

Block of tetrodotoxin-resistant Na+ channel pore by multivalent cations: gating modification and Na+ flow dependence

Tetrodotoxin-resistant (TTX-R) Na(+) channels are much less susceptible to external TTX but more susceptible to external Cd(2+) block than tetrodotoxin-sensitive (TTX-S) Na(+) channels. Both TTX and Cd(2+) seem to block the channel near the "DEKA" ring, which is probably part of a multi-ion single-file region adjacent to the external pore mouth and is involved in the selectivity filter of the channel. In this study we demonstrate that other multivalent transitional metal ions such as La(3+), Zn(2+), Ni(2+), Co(2+), and Mn(2+) also block the TTX-R channels in dorsal root ganglion neurons. Just like Cd(2+), the blocking effect has little intrinsic voltage dependence, but is profoundly influenced by Na(+) flow. The apparent dissociation constants of the blocking ions are always significantly smaller in inward Na(+) currents than those in outward Na(+) current, signaling exit of the blocker along with the Na(+) flow and a high internal energy barrier for "permeation" of these multivalent blocking ions through the pore. Most interestingly, the activation and especially the inactivation kinetics are slowed by the blocking ions. Moreover, the gating changes induced by the same concentration of a blocking ion are evidently different in different directions of Na(+) current flow, but can always be correlated with the extent of pore block. Further quantitative analyses indicate that the apparent slowing of channel activation is chiefly ascribable to Na(+) flow-dependent unblocking of the bound La(3+) from the open Na(+) channel, whereas channel inactivation cannot happen with any discernible speed in the La(3+)-blocked channel. Thus, the selectivity filter of Na(+) channel is probably contiguous to a single-file multi-ion region at the external pore mouth, a region itself being nonselective in terms of significant binding of different multivalent cations. This region is "open" to the external solution even if the channel is "closed" ("deactivated"), but undergoes imperative conformational changes during the gating (especially the inactivation) process of the channel.     

Halogen substituents on the aromatic moiety of the tetracaine scaffold improve potency of cyclic nucleotide-gated channel block

A series of new tetracaine derivatives with substituents on the aromatic ring was prepared and evaluated for block of retinal rod cyclic nucleotide-gated (CNG) channels. Aromatic substitutions had little effect starting with the basic tetracaine scaffold, but electron-withdrawing substituents significantly improved the blocking potency of an octyl-tail derivative of tetracaine. In particular, halogen substitutions at either the 2- or 3-position on the ring resulted in compounds that were up to eight-fold more potent than the parent octyl-tail derivative and up to 50-fold more potent than tetracaine.     

Ion-Ion interactions at the selectivity filter. Evidence from K(+)-dependent modulation of tetraethylammonium efficacy in Kv2.1 potassium channels

In the Kv2.1 potassium channel, binding of K(+) to a high-affinity site associated with the selectivity filter modulates channel sensitivity to external TEA. In channels carrying Na(+) current, K(+) interacts with the TEA modulation site at concentrations 相似文献   

Na+ block and permeation in a K+ channel of known structure

The effects of intracellular Na(+) were studied on K(+) and Rb(+) currents through single KcsA channels. At low voltage, Na(+) produces voltage-dependent block, which becomes relieved at high voltage by a "punchthrough" mechanism representing Na(+) escaping from its blocking site through the selectivity filter. The Na(+) blocking site is located in the wide, hydrated vestibule, and it displays unexpected selectivity for K(+) and Rb(+) against Na(+). The voltage dependence of Na(+) block reflects coordinated movements of the blocker with permeant ions in the selectivity filter.     

Ion solvation and structural stability in a sodium channel investigated by molecular dynamics calculations

The stability and ion binding properties of the homo-tetrameric pore domain of a prokaryotic, voltage-gated sodium channel are studied by extensive all-atom molecular dynamics simulations, with the channel protein being embedded in a fully hydrated lipid bilayer. It is found that Na(+) ion presents in a mostly hydrated state inside the wide pore of the selectivity filter of the sodium channel, in sharp contrast to the nearly fully dehydrated state for K(+) ions in potassium channels. Our results also indicate that Na(+) ions make contact with only one or two out of the four polypeptide chains forming the selectivity filter, and surprisingly, the selectivity filter exhibits robust stability for various initial ion configurations even in the absence of ions. These findings are quite different from those in potassium channels. Furthermore, an electric field above 0.5V/nm is suggested to be able to induce Na(+) permeation through the selectivity filter.     

Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.     

K(+) versus Na(+) ions in a K channel selectivity filter: a simulation study

Molecular dynamics simulations of a bacterial potassium channel (KcsA) embedded in a phospholipid bilayer reveal significant differences in interactions of the selectivity filter with K(+) compared with Na(+) ions. K(+) ions and water molecules within the filter undergo concerted single-file motion in which they translocate between adjacent sites within the filter on a nanosecond timescale. In contrast, Na(+) ions remain bound to sites within the filter and do not exhibit translocation on a nanosecond timescale. Furthermore, entry of a K(+) ion into the filter from the extracellular mouth is observed, whereas this does not occur for a Na(+) ion. Whereas K(+) ions prefer to sit within a cage of eight oxygen atoms of the filter, Na(+) ions prefer to interact with a ring of four oxygen atoms plus two water molecules. These differences in interactions in the selectivity filter may contribute to the selectivity of KcsA for K(+) ions (in addition to the differences in dehydration energy between K(+) and Na(+)) and the block of KcsA by internal Na(+) ions. In our simulations the selectivity filter exhibits significant flexibility in response to changes in ion/protein interactions, with a somewhat greater distortion induced by Na(+) than by K(+) ions.     

Dequalinium: a novel,high-affinity blocker of CNGA1 channels

Cyclic nucleotide-gated (CNG) channels have been shown to be blocked by diltiazem, tetracaine, polyamines, toxins, divalent cations, and other compounds. Dequalinium is an organic divalent cation which suppresses the rat small conductance Ca(2+)-activated K(+) channel 2 (rSK2) and the activity of protein kinase C. In this study, we have tested the ability of dequalinium to block CNGA1 channels and heteromeric CNGA1+CNGB1 channels. When applied to the intracellular side of inside-out excised patches from Xenopus oocytes, dequalinium blocks CNGA1 channels with a K(1/2) approximately 190 nM and CNGA1+CNGB1 channels with a K(1/2) approximately 385 nM, at 0 mV. This block occurs in a state-independent fashion, and is voltage dependent with a zdelta approximately 1. Our data also demonstrate that dequalinium interacts with the permeant ion probably because it occupies a binding site in the ion conducting pathway. Dequalinium applied to the extracellular surface also produced block, but with a voltage dependence that suggests it crosses the membrane to block from the inside. We also show that at the single-channel level, dequalinium is a slow blocker that does not change the unitary conductance of CNGA1 channels. Thus, dequalinium should be a useful tool for studying permeation and gating properties of CNG channels.     

Ion selectivity and current saturation in inward-rectifier K+ channels

We investigated the features of the inward-rectifier K channel Kir1.1 (ROMK) that underlie the saturation of currents through these channels as a function of permeant ion concentration. We compared values of maximal currents and apparent K(m) for three permeant ions: K(+), Rb(+), and NH(4)(+). Compared with K(+) (i(max) = 4.6 pA and K(m) = 10 mM at -100 mV), Rb(+) had a lower permeability, a lower i(max) (1.8 pA), and a higher K(m) (26 mM). For NH(4)(+), the permeability was reduced more with smaller changes in i(max) (3.7 pA) and K(m) (16 mM). We assessed the role of a site near the outer mouth of channel in the saturation process. This site could be occupied by either permeant ions or low-affinity blocking ions such as Na(+), Li(+), Mg(2+), and Ca(2+) with similar voltage dependence (apparent valence, 0.15-0.20). It prefers Mg(2+) over Ca(2+) and has a monovalent cation selectivity, based on the ability to displace Mg(2+), of K(+) > Li(+) ～ Na(+) > Rb(+) ～ NH(4)(+). Conversely, in the presence of Mg(2+), the K(m) for K(+) conductance was substantially increased. The ability of Mg(2+) to block the channels was reduced when four negatively charged amino acids in the extracellular domain of the channel were mutated to neutral residues. The apparent K(m) for K(+) conduction was unchanged by these mutations under control conditions but became sensitive to the presence of external negative charges when residual divalent cations were chelated with EDTA. The results suggest that a binding site in the outer mouth of the pore controls current saturation. Permeability is more affected by interactions with other sites within the selectivity filter. Most features of permeation (and block) could be simulated by a five-state kinetic model of ion movement through the channel.     

Common molecular determinants of flecainide and lidocaine block of heart Na+ channels: evidence from experiments with neutral and quaternary flecainide analogues

Flecainide (pKa 9.3, 99% charged at pH 7.4) and lidocaine (pKa 7.6-8.0, approximately 50% neutral at pH 7.4) have similar structures but markedly different effects on Na(+) channel activity. Both drugs cause well-characterized use-dependent block (UDB) of Na(+) channels due to stabilization of the inactivated state, but flecainide requires that channels first open before block develops, whereas lidocaine is believed to bind directly to the inactivated state. To test whether the charge on flecainide might determine its state specificity of Na(+) channel blockade, we developed two flecainide analogues, NU-FL (pKa 6.4), that is 90% neutral at pH 7.4, and a quaternary flecainide analogue, QX-FL, that is fully charged at physiological pH. We examined the effects of flecainide, NU-FL, QX-FL, and lidocaine on human cardiac Na(+) channels expressed in human embryonic kidney (HEK) 293 cells. At physiological pH, NU-FL, like lidocaine but not flecainide, interacts preferentially with inactivated channels without prerequisite channel opening, and causes minimal UDB. We find that UDB develops predominantly by the charged form of flecainide as evidenced by investigation of QX-FL at physiological pH and NU-FL investigated over a more acidic pH range where its charged fraction is increased. QX-FL is a potent blocker of channels when applied from inside the cell, but acts very weakly with external application. UDB by QX-FL, like flecainide, develops only after channels open. Once blocked, channels recover very slowly from QX-FL block, apparently without requisite channel opening. Our data strongly suggest that it is the difference in degree of ionization (pKa) between lidocaine and flecainide, rather than gross structural features, that determines distinction in block of cardiac Na(+) channels. The data also suggest that the two drugs share a common receptor but, consistent with the modulated receptor hypothesis, reach this receptor by distinct routes dictated by the degree of ionization of the drug molecules.     

Site-specific mutations in a minimal voltage-dependent K+ channel alter ion selectivity and open-channel block

MinK is a small membrane protein of 130 amino acids with a single potential membrane-spanning alpha-helical domain. Its expression in Xenopus oocytes induces voltage-dependent, K(+)-selective channels. Using site-directed mutagenesis of a synthetic gene, we have identified residues in the hydrophobic region of minK that influence both ion selectivity and open-channel block. Single amino acid changes increase the channel's relative permeability for NH4+ and Cs+ without affecting its ability to exclude Na+ and Li+. Blockade by two common K+ channel pore blockers, tetraethylammonium and Cs+, was also modified. These results suggest that an ion selectivity region and binding sites for the pore blockers within the conduction pathway have been modified. We conclude that the gene encoding minK is a structural gene for a K+ channel protein.     

Sp-tetraKCNG: A novel cyclic nucleotide gated K(+) channel

The sequence of a novel cGMP-regulated, tetrameric, K(+) selective channel (Sp-tetraKCNG) was discovered in the sea urchin Strongylocentrotus purpuratus. The Sp-tetraKCNG is a single polypeptide made of four KCNG domains similar to voltage-dependent Na(+) and Ca(2+) channels. Each KCNG domain has six transmembrane segments (S1-S6), the ion pore having the K(+) selectivity signature GYGD and a cyclic nucleotide-binding domain (CNBD). This novel channel is evolutionary located between K(+)-selective and voltage-dependent EAG channels and voltage-independent cationic CNG channels. Bilayer reconstitutions demonstrate such a cGMP-regulated K(+) selective channel in sea urchin spermatozoa.