Protein kinase regulation of a cloned epithelial Na+ channel

We examined the regulation of a cloned epithelial Na+ channel (alpha beta gamma-rENaC) by protein kinase A (PKA) and protein kinase C (PKC). Experiments were performed in Xenopus oocytes and in planar lipid bilayers. At a holding potential of -100 mV, amiloride-sensitive current averaged -1,279 +/- 111 nA (n = 7) in alpha beta gamma-rENaC- expressing oocytes. Currents in water-injected oocytes were essentially unresponsive to 10 microM amiloride. A 1-h stimulation of PKC with 100 nM of PMA inhibited whole-cell currents in Xenopus oocytes to 17.1 +/- 1.8, and 22.1 +/- 2.6% of control (n = 7), at holding potentials of - 100 and +40 mV, respectively. Direct injection of purified PKC resulted in similar inhibition to that observed with PMA. Additionally, the inactive phorbol ester, phorbol-12-myristate-13-acetate, 4-O-methyl, was without effect on alpha beta gamma-rENaC currents. Pretreatment with the microtubule inhibitor colchicine (100 microM) did not modify the inhibitory effect of PMA; however, pretreatment with 20 microM cytochalasin B decreased the inhibitory action of PMA to < 20% of that previously observed. In vitro-synthesized alpha beta gamma-rENaC formed an amiloride-sensitive Na(+)-selective channel when incorporated into planar lipid bilayers. Addition of PKC, diacyl-glycerol, and Mg-ATP to the side opposite that which amiloride blocked, decreased the channel''s open probability (Po) from 0.44 +/- 0.06 to 0.13 +/- 0.03 (n = 9). To study the effects of PKA on alpha beta gamma-rENaC expressed in Xenopus oocytes, cAMP levels were elevated with 10 microM forskolin and 1 mM isobutyl-methyl-xanthine. This cAMP-elevating cocktail did not cause any stimulation of alpha beta gamma-rENaC currents in either the inward or outward directions. This lack of activation was also observed in oocytes preinhibited with PMA and in oocytes pretreated with cytochalasin B and PMA. Neither alpha-rENaC nor alpha beta gamma-rENaC incorporated into planar lipid bilayers could be activated with PKA and Mg-ATP added to either side of the membrane, as Po remained at 0.63 +/- 0.06 (n = 7) and 0.45 +/- 0.05 (n = 9), respectively. We conclude that: alpha beta gamma-rENaC is inhibited by PKC, and that alpha beta gamma- rENaC is not activated by PKA.     

The NH(2) terminus of the epithelial sodium channel contains an endocytic motif.

An epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. To elucidate the function of the cytoplasmic, NH(2) terminus of rat ENaC (rENaC) subunits, a series of mutant cDNAs was constructed and the cRNAs for all three subunits were expressed in Xenopus oocytes. Amiloride-sensitive Na(+) currents (I(Na)) were measured by the two-electrode voltage clamp technique. Deletion of the cytoplasmic, NH(2) terminus of alpha (Delta2-109), beta (Delta2-49), or gamma-rENaC (Delta2-53) dramatically reduced I(Na). A series of progressive, NH(2)-terminal deletions of alpha-rENaC were constructed to identify motifs that regulate I(Na). Deletion of amino acids 2-46 had no effect on I(Na): however, deletion of amino acids 2-51, 2-55, 2-58, and 2-67 increased I(Na) by approximately 4-fold. By contrast, deletion of amino acids 2-79, 2-89, 2-100, and 2-109 eliminated I(Na). To evaluate the mechanism whereby Delta2-67-alpha-rENaC increased I(Na), single channels were evaluated by patch clamp. The single-channel conductance and open probability of alpha,beta,gamma-rENaC and Delta2-67-alpha,beta,gamma-rENaC were similar. However, the number of active channels in the membrane increased from 6 +/- 1 channels per patch with alpha,beta,gamma-rENaC to 11 +/- 1 channels per patch with Delta2-67-alpha,beta,gamma-rENaC. Laser scanning confocal microscopy confirmed that there were more Delta2-67-alpha,beta, gamma-rENaC channels in the plasma membrane than alpha,beta, gamma-rENaC channels. Deletion of amino acids 2-67 in alpha-rENaC reduced the endocytic retrieval of channels from the plasma membrane and increased the half-life of the channel in the membrane from 1.1 +/- 0.2 to 3.5 +/- 1.1 h. We conclude that the cytoplasmic, NH(2) terminus of alpha-, beta-, and gamma-rENaC is required for channel activity. The cytoplasmic, NH(2) terminus of alpha-rENaC contains two key motifs. One motif regulates the endocytic retrieval of the channel from the plasma membrane. The second motif is required for channel activity.     

The beta subunit increases the Ca2+ sensitivity of large conductance Ca2+-activated potassium channels by retaining the gating in the bursting states

Coexpression of the beta subunit (KV,Cabeta) with the alpha subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the beta subunit increased open probability (Po) by increasing burst duration 20-100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the beta subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the beta subunit does not act by increasing all the Ca2+ binding rates proportionally. The beta subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the beta subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the beta subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.     

The epithelial Na+ channel is inhibited by a peptide derived from proteolytic processing of its alpha subunit

Epithelial sodium channels (ENaCs) mediate Na(+) entry across the apical membrane of high resistance epithelia that line the distal nephron, airway and alveoli, and distal colon. These channels are composed of three homologous subunits, termed alpha, beta, and gamma, which have intracellular amino and carboxyl termini and two membrane-spanning domains connected by large extracellular loops. Maturation of ENaC subunits involves furin-dependent cleavage of the extracellular loops at two sites within the alpha subunit and at a single site within the gamma subunit. The alpha subunits must be cleaved twice, immediately following Arg-205 and Arg-231, in order for channels to be fully active. Channels lacking alpha subunit cleavage are inactive with a very low open probability. In contrast, channels lacking both alpha subunit cleavage and the tract alphaAsp-206-Arg-231 are active when expressed in oocytes, suggesting that alphaAsp-206-Arg-231 functions as an inhibitor that stabilizes the channel in the closed conformation. A synthetic 26-mer peptide (alpha-26), corresponding to alphaAsp-206-Arg-231, reversibly inhibits wild-type mouse ENaCs expressed in Xenopus oocytes, as well as endogenous Na(+) channels expressed in either a mouse collecting duct cell line or primary cultures of human airway epithelial cells. The IC(50) for amiloride block of ENaC was not affected by the presence of alpha-26, indicating that alpha-26 does not bind to or interact with the amiloride binding site. Substitution of Arg residues within alpha-26 with Glu, or substitution of Pro residues with Ala, significantly reduced the efficacy of alpha-26. The peptide inhibits ENaC by reducing channel open probability. Our results suggest that proteolysis of the alpha subunit activates ENaC by disassociating an inhibitory domain (alphaAsp-206-Arg-231) from its effector site within the channel complex.     

Differential activation by Ca2+, ATP and caffeine of cardiac and skeletal muscle ryanodine receptors after block by Mg2+

The block of rabbit skeletal ryanodine receptors (RyR1) and dog heart RyR2 by cytosolic [Mg2+], and its reversal by agonists Ca2+, ATP and caffeine was studied in planar bilayers. Mg2+ effects were tested at submaximal activating [Ca2+] (5 microM). Approximately one third of the RyR1s had low open probability ("LA channels") in the absence of Mg2+. All other RyR1s displayed higher activity ("HA channels"). Cytosolic Mg2+ (1 mM) blocked individual RyR1 channels to varying degrees (32 to 100%). LA channels had residual P(o) <0.005 in 1 mM Mg2+ and reactivated poorly with [Ca2+] (100 microM), caffeine (5 mM), or ATP (4 mM; all at constant 1 mM Mg2+). HA channels had variable activity in Mg2+ and variable degree of recovery from Mg2+ block with Ca2+, caffeine or ATP application. Nearly all cardiac RyR2s displayed high activity in 5 microM [Ca2+]. They also had variable sensitivity to Mg2+. However, the RyR2s consistently recovered from Mg2+ block with 100 microM [Ca2+] or caffeine application, but not when ATP was added. Thus, at physiological [Mg2+], RyR2s behaved as relatively homogeneous Ca2+/caffeine-gated HA channels. In contrast, RyR1s displayed functional heterogeneity that arises from differential modulatory actions of Ca2+ and ATP. These differences between RyR1 and RyR2 function may reflect their respective roles in muscle physiology and excitation-contraction coupling.     

Regulation by Na+ and Ca2+ of renal epithelial Na+ channels reconstituted into planar lipid bilayers

Purified bovine renal epithelial Na+ channels when reconstituted into planar lipid bilayers displayed a specific orientation when the membrane was clamped to -40 mV (cis-side) during incorporation. The trans-facing portion of the channel was extracellular (i.e., amiloride- sensitive), whereas the cis-facing side was intracellular (i.e., protein kinase A-sensitive). Single channels had a main state unitary conductance of 40 pS and displayed two subconductive states each of 12- 13 pS, or one of 12-13 pS and the second of 24-26 pS. Elevation of the [Na+] gradient from the trans-side increased single-channel open probability (Po) only when the cis-side was bathed with a solution containing low [Na+] (< 30 mM) and 10-100 microM [Ca2+]. Under these conditions, Po saturated with increasing [Na+]trans. Buffering of the cis compartment [Ca2+] to nearly zero (< 1 nM) with 10 mM EGTA increased the initial level of channel activity (Po = 0.12 +/- 0.02 vs 0.02 +/- 0.01 in control), but markedly reduced the influence of both cis- and trans-[Na+] on Po. Elevating [Ca2+]cis at constant [Na+] resulted in inhibition of channel activity with an apparent [KiCa2+] of 10-100 microM. Protein kinase C-induced phosphorylation shifted the dependence of channel Po on [Ca2+]cis to 1-3 microM at stationary [Na+]. The direct modulation of single-channel Po by Na+ and Ca2+ demonstrates that the gating of amiloride-sensitive Na2+ channels is indeed dependent upon the specific ionic environment surrounding the channels.     

Divalent cation selectivity for external block of voltage-dependent Na+ channels prolonged by batrachotoxin. Zn2+ induces discrete substates in cardiac Na+ channels

The mechanism of block of voltage-dependent Na+ channels by extracellular divalent cations was investigated in a quantitative comparison of two distinct Na+ channel subtypes incorporated into planar bilayers in the presence of batrachotoxin. External Ca2+ and other divalent cations induced a fast voltage-dependent block observed as a reduction in unitary current for tetrodotoxin-sensitive Na+ channels of rat skeletal muscle and tetrodotoxin-insensitive Na+ channels of canine heart ventricular muscle. Using a simple model of voltage-dependent binding to a single site, these two distinct Na+ channel subtypes exhibited virtually the same affinity and voltage dependence for fast block by Ca2+ and a number of other divalent cations. This group of divalent cations exhibited an affinity sequence of Co congruent to Ni greater than Mn greater than Ca greater than Mg greater than Sr greater than Ba, following an inverse correlation between binding affinity and ionic radius. The voltage dependence of fast Ca2+ block was essentially independent of CaCl2 concentration; however, at constant voltage the Ca2+ concentration dependence of fast block deviated from a Langmuir isotherm in the manner expected for an effect of negative surface charge. Titration curves for fast Ca2+ block were fit to a simplified model based on a single Ca2+ binding site and the Gouy-Chapman theory of surface charge. This model gave similar estimates of negative surface charge density in the vicinity of the Ca2+ blocking site for muscle and heart Na+ channels. In contrast to other divalent cations listed above, Cd2+ and Zn2+ are more potent blockers of heart Na+ channels than muscle Na+ channels. Cd2+ induced a fast, voltage-dependent block in both Na+ channel subtypes with a 46-fold higher affinity at 0 mV for heart (KB = 0.37 mM) vs. muscle (KB = 17 mM). Zn2+ induced a fast, voltage-dependent block of muscle Na+ channels with low affinity (KB = 7.5 mM at 0 mV). In contrast, micromolar Zn2+ induced brief closures of heart Na+ channels that were resolved as discrete substate events at the single-channel level with an apparent blocking affinity of KB = 0.067 mM at 0 mV, or 110-fold higher affinity for Zn2+ compared with the muscle channel. High-affinity block of the heart channel by Cd2+ and Zn2+ exhibited approximately the same voltage dependence (e-fold per 60 mV) as low affinity block of the muscle subtype (e-fold per 54 mV), suggesting that the block occurs at structurally analogous sites in the two Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength

Charybdotoxin (CTX), a small, basic protein from scorpion venom, strongly inhibits the conduction of K ions through high-conductance, Ca2+-activated K+ channels. The interaction of CTX with Ca2+-activated K+ channels from rat skeletal muscle plasma membranes was studied by inserting single channels into uncharged planar phospholipid bilayers. CTX blocks K+ conduction by binding to the external side of the channel, with an apparent dissociation constant of approximately 10 nM at physiological ionic strength. The dwell-time distributions of both blocked and unblocked states are single-exponential. The toxin association rate varies linearly with the CTX concentration, and the dissociation rate is independent of it. CTX is competent to block both open and closed channels; the association rate is sevenfold faster for the open channel, while the dissociation rate is the same for both channel conformations. Membrane depolarization enhances the CTX dissociation rate e-fold/28 mV; if the channel's open probability is maintained constant as voltage varies, then the toxin association rate is voltage independent. Increasing the external solution ionic strength from 20 to 300 mM (with K+, Na+, or arginine+) reduces the association rate by two orders of magnitude, with little effect on the dissociation rate. We conclude that CTX binding to the Ca2+-activated K+ channel is a bimolecular process, and that the CTX interaction senses both voltage and the channel's conformational state. We further propose that a region of fixed negative charge exists near the channel's CTX-binding site.     

The beta1 subunit enhances oxidative regulation of large-conductance calcium-activated K+ channels

Oxidative stress may alter the functions of many proteins including the Slo1 large conductance calcium-activated potassium channel (BKCa). Previous results demonstrated that in the virtual absence of Ca2+, the oxidant chloramine-T (Ch-T), without the involvement of cysteine oxidation, increases the open probability and slows the deactivation of BKCa channels formed by human Slo1 (hSlo1) alpha subunits alone. Because native BKCa channel complexes may include the auxiliary subunit beta1, we investigated whether beta1 influences the oxidative regulation of hSlo1. Oxidation by Ch-T with beta1 present shifted the half-activation voltage much further in the hyperpolarizing direction (-75 mV) as compared with that with alpha alone (-30 mV). This shift was eliminated in the presence of high [Ca2+]i, but the increase in open probability in the virtual absence of Ca2+ remained significant at physiologically relevant voltages. Furthermore, the slowing of channel deactivation after oxidation was even more dramatic in the presence of beta1. Oxidation of cysteine and methionine residues within beta1 was not involved in these potentiated effects because expression of mutant beta1 subunits lacking cysteine or methionine residues produced results similar to those with wild-type beta1. Unlike the results with alpha alone, oxidation by Ch-T caused a significant acceleration of channel activation only when beta1 was present. The beta1 M177 mutation disrupted normal channel activation and prevented the Ch-T-induced acceleration of activation. Overall, the functional effects of oxidation of the hSlo1 pore-forming alpha subunit are greatly amplified by the presence of beta1, which leads to the additional increase in channel open probability and the slowing of deactivation. Furthermore, M177 within beta1 is a critical structural determinant of channel activation and oxidative sensitivity. Together, the oxidized BKCa channel complex with beta1 has a considerable chance of being open within the physiological voltage range even at low [Ca2+]i.     

pH alterations "reset" Ca2+ sensitivity of brain Na+ channel 2, a degenerin/epithelial Na+ ion channel, in planar lipid bilayers

Members of the degenerin/epithelial Na(+) channel superfamily of ion channels subserve many functions, ranging from whole body sodium handling to mechanoelectrical transduction. We studied brain Na(+) channel 2 (BNaC-2) in planar lipid bilayers to examine its single channel properties and regulation by Ca(2+). Upon incorporation of vesicles made from membranes of oocytes expressing either wild-type (WT) BNaC-2 or BNaC-2 with a gain-of-function (GF) point mutation (G433F), functional channels with different properties were obtained. WT BNaC-2 resided in a closed state with short openings, whereas GF BNaC-2 was constitutively activated; a decrease in the pH in the trans compartment of the bilayer activated WT BNaC-2 and decreased its permeability for Na(+) over K(+). Moreover, these maneuvers made the WT channel more resistant to amiloride. In contrast, GF BNaC-2 did not respond to a decrease in pH, and its amiloride sensitivity and selectivity for Na(+) over K(+) were unaffected by this pH change. Buffering the bathing solutions with EGTA to reduce the free [Ca(2+)] to <10 nm increased WT single channel open probability 10-fold, but not that of GF BNaC-2. Ca(2+) blocked both WT and GF BNaC-2 in a dose- and voltage-dependent fashion; single channel conductances were unchanged. A drop in pH reduced the ability of Ca(2+) to inhibit these channels. These results show that BNaC-2 is an amiloride-sensitive sodium channel and suggest that pH activation of these channels could be, in part, a consequence of H(+) "interference" with channel regulation by Ca(2+).     

Cystic fibrosis transmembrane conductance regulator. Physical basis for lyotropic anion selectivity patterns

The selectivity of Ca2+ over Na+ is approximately 3.3-fold larger in cGMP-gated channels of cone photoreceptors than in those of rods when measured under saturating cGMP concentrations, where the probability of channel opening is 85-90%. Under physiological conditions, however, the probability of opening of the cGMP-gated channels ranges from its largest value in darkness of 1-5% to essentially zero under continuous, bright illumination. We investigated the ion selectivity of cGMP-gated channels as a function of cyclic nucleotide concentration in membrane patches detached from the outer segments of rod and cone photoreceptors and have found that ion selectivity is linked to gating. We determined ion selectivity relative to Na+ (PX/PNa) from the value of reversal potentials measured under ion concentration gradients. The selectivity for Ca2+ over Na+ increases continuously as the probability of channel opening rises. The dependence of PCa/PNa on cGMP concentration, in both rods and cones, is well described by the same Hill function that describes the cGMP dependence of current amplitude. At the cytoplasmic cGMP concentrations expected in dark-adapted intact photoreceptors, PCa/PNa in cone channels is approximately 7.4-fold greater than that in rods. The linkage between selectivity and gating is specific for divalent cations. The selectivity of Ca2+ and Sr2+ changes with cGMP concentration, but the selectivity of inorganic monovalent cations, Cs+ and NH4+, and organic cations, methylammonium+ and dimethylammonium+, is invariant with cGMP. Cyclic nucleotide-gated channels in rod photoreceptors are heteromeric assemblies of alpha and beta subunits. The maximal PCa/PNa of channels formed from alpha subunits of bovine rod channels is less than that of heteromeric channels formed from alpha and beta subunits. In addition, Ca2+ is a more effective blocker of channels formed by alpha subunits than of channels formed by alpha and beta subunits. The cGMP-dependent shift in divalent cation selectivity is a property of alphabeta channels and not of channels formed from alpha subunits alone.     

Effective gating charges per channel in voltage-dependent K+ and Ca2+ channels

In voltage-dependent ion channels, the gating of the channels is determined by the movement of the voltage sensor. This movement reflects the rearrangement of the protein in response to a voltage stimulus, and it can be thought of as a net displacement of elementary charges (e0) through the membrane (z: effective number of elementary charges). In this paper, we measured z in Shaker IR (inactivation removed) K+ channels, neuronal alpha 1E and alpha 1A, and cardiac alpha 1C Ca2+ channels using two methods: (a) limiting slope analysis of the conductance-voltage relationship and (b) variance analysis, to evaluate the number of active channels in a patch, combined with the measurement of charge movement in the same patch. We found that in Shaker IR K+ channels the two methods agreed with a z congruent to 13. This suggests that all the channels that gate can open and that all the measured charge is coupled to pore opening in a strictly sequential kinetic model. For all Ca2+ channels the limiting slope method gave consistent results regardless of the presence or type of beta subunit tested (z = 8.6). However, as seen with alpha 1E, the variance analysis gave different results depending on the beta subunit used. alpha 1E and alpha 1E beta 1a gave higher z values (z = 14.77 and z = 15.13 respectively) than alpha 1E beta 2a (z = 9.50, which is similar to the limiting slope results). Both the beta 1a and beta 2a subunits, coexpressed with alpha 1E Ca2+ channels facilitated channel opening by shifting the activation curve to more negative potentials, but only the beta 2a subunit increased the maximum open probability. The higher z using variance analysis in alpha 1E and alpha 1E beta 1a can be explained by a set of charges not coupled to pore opening. This set of charges moves in transitions leading to nulls thus not contributing to the ionic current fluctuations but eliciting gating currents. Coexpression of the beta 2a subunit would minimize the fraction of nulls leading to the correct estimation of the number of channels and z.     

Single Na+ channels activated by veratridine and batrachotoxin

Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded.     

Functional coupling of the beta(1) subunit to the large conductance Ca(2+)-activated K(+) channel in the absence of Ca(2+). Increased Ca(2+) sensitivity from a Ca(2+)-independent mechanism

Coexpression of the beta(1) subunit with the alpha subunit (mSlo) of BK channels increases the apparent Ca(2+) sensitivity of the channel. This study investigates whether the mechanism underlying the increased Ca(2+) sensitivity requires Ca(2+), by comparing the gating in 0 Ca(2+)(i) of BK channels composed of alpha subunits to those composed of alpha+beta(1) subunits. The beta(1) subunit increased burst duration approximately 20-fold and the duration of gaps between bursts approximately 3-fold, giving an approximately 10-fold increase in open probability (P(o)) in 0 Ca(2+)(i). The effect of the beta(1) subunit on increasing burst duration was little changed over a wide range of P(o) achieved by varying either Ca(2+)(i) or depolarization. The effect of the beta(1) subunit on increasing the durations of the gaps between bursts in 0 Ca(2+)(i) was preserved over a range of voltage, but was switched off as Ca(2+)(i) was increased into the activation range. The Ca(2+)-independent, beta(1) subunit-induced increase in burst duration accounted for 80% of the leftward shift in the P(o) vs. Ca(2+)(i) curve that reflects the increased Ca(2+) sensitivity induced by the beta(1) subunit. The Ca(2+)-dependent effect of the beta(1) subunit on the gaps between bursts accounted for the remaining 20% of the leftward shift. Our observation that the major effects of the beta(1) subunit are independent of Ca(2+)(i) suggests that the beta(1) subunit mainly alters the energy barriers of Ca(2+)-independent transitions. The changes in gating induced by the beta(1) subunit differ from those induced by depolarization, as increasing P(o) by depolarization or by the beta(1) subunit gave different gating kinetics. The complex gating kinetics for both alpha and alpha+beta(1) channels in 0 Ca(2+)(i) arise from transitions among two to three open and three to five closed states and are inconsistent with Monod-Wyman-Changeux type models, which predict gating among only one open and one closed state in 0 Ca(2+)(i).     

Unitary Ca2+ current through mammalian cardiac and amphibian skeletal muscle ryanodine receptor Channels under near-physiological ionic conditions

Ryanodine receptor (RyR) channels from mammalian cardiac and amphibian skeletal muscle were incorporated into planar lipid bilayers. Unitary Ca2+ currents in the SR lumen-to-cytosol direction were recorded at 0 mV in the presence of caffeine (to minimize gating fluctuations). Currents measured with 20 mM lumenal Ca2+ as exclusive charge carrier were 4.00 and 4.07 pA, respectively, and not significantly different. Currents recorded at 1-30 mM lumenal Ca2+ concentrations were attenuated by physiological [K+] (150 mM) and [Mg2+] (1 mM), in the same proportion (approximately 55%) in mammalian and amphibian channels. Two amplitudes, differing by approximately 35%, were found in amphibian channel studies, probably corresponding to alpha and beta RyR isoforms. In physiological [Mg2+], [K+], and lumenal [Ca2+] (1 mM), the Ca2+ current was just less than 0.5 pA. Comparison of this value with the Ca2+ flux underlying Ca2+ sparks suggests that sparks in mammalian cardiac and amphibian skeletal muscles are generated by opening of multiple RyR channels. Further, symmetric high concentrations of Mg2+ substantially reduced the current carried by 10 mM Ca2+ (approximately 40% at 10 mM Mg2+), suggesting that high Mg2+ may make sparks smaller by both inhibiting RyR gating and reducing unitary current.     

Cystic fibrosis transmembrane conductance regulator inhibits epithelial Na+ channels carrying Liddle's syndrome mutations.

Epithelial Na+ channels (ENaC) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) upon activation by protein kinase A. It is, however, still unclear how CFTR regulates the activity of ENaC. In the present study we examined whether CFTR interacts with ENaC by interfering with the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various C-terminal mutations were introduced into the three alpha-, beta-, and gamma-subunits of the rat epithelial Na+ channel, thereby eliminating PY motifs, which are important binding domains for the ubiquitin ligase Nedd4. When expressed in Xenopus oocytes, most of the ENaC stop (alpha-H647X, beta-P565X, gamma-S608X) or point (alpha-P671A, beta-Y618A, gamma-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type alpha,beta,gamma-rENaC. However, ENaC currents formed by either of the mutant alpha-, beta-, or gamma-subunits were inhibited during activation of CFTR by forskolin (10 micromol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced alpha,beta,gamma-rENaC whole cell Na+ conductance but did not interfere with inhibition of ENaC by CFTR. Another mutant, beta-T592M,T593A-ENaC, also showed enhanced Na+ currents, which were down-regulated by CFTR. Moreover, activation of ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that regulation of ENaC by CFTR is distal to other regulatory limbs and does not involve Nedd4-dependent ubiquitination.     

Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by palmitoyl carnitine.

Studies of [3H]ryanodine binding, 45Ca2+ efflux, and single channel recordings in planar bilayers indicated that the fatty acid metabolite palmitoyl carnitine produced a direct stimulation of the Ca2+ release channel (ryanodine receptor) of rabbit and pig skeletal muscle junctional sarcoplasmic reticulum. At a concentration of 50 microM, palmitoyl carnitine (a) stimulated [3H]ryanodine binding 1.6-fold in a competitive manner at all pCa in the range 6 to 3; (b) released approximately 65% (30 nmol) of passively loaded 45Ca2+/mg protein; and (c) increased 7-fold the open probability of Ca2+ release channels incorporated into planar bilayers. Neither carnitine nor palmitic acid could reproduce the effect of palmitoyl carnitine on [3H]ryanodine binding, 45Ca2+ release, or channel open probability. 45Ca2+ release was induced by several long-chain acyl carnitines (C14, C16, C18) and acyl coenzyme A derivatives (C12, C14, C16), but not by the short-chain derivative C8 or by free saturated fatty acids of chain length C8 to C18, at room temperature or 36 degrees C. This newly identified interaction of esterified fatty acids and ryanodine receptors may represent a pathway by which metabolism of skeletal muscle could influence intracellular Ca2+ and may be responsible for the pathophysiology of disorders of beta-oxidation such as carnitine palmitoyl transferase II deficiency.     

Lipid surface charge does not influence conductance or calcium block of single sodium channels in planar bilayers.

We have studied the effects of membrane surface charge on Na+ ion permeation and Ca2+ block in single, batrachotoxin-activated Na channels from rat brain, incorporated into planar lipid bilayers. In phospholipid membranes with no net charge (phosphatidylethanolamine, PE), at low divalent cation concentrations (approximately 100 microM Mg2+), the single channel current-voltage relation was linear and the single channel conductance saturated with increasing [Na+] and ionic strength, reaching a maximum (gamma max) of 31.8 pS, with an apparent dissociation constant (K0.5) of 40.5 mM. The data could be approximated by a rectangular hyperbola. In negatively charged bilayers (70% phosphatidylserine, PS; 30% PE) slightly larger conductances were observed at each concentration, but the hyperbolic form of the conductance-concentration relation was retained (gamma max = 32.9 pS and K0.5 = 31.5 mM) without any preferential increase in conductance at lower ionic strengths. Symmetrical application of Ca2+ caused a voltage-dependent block of the single channel current, with the block being greater at negative potentials. For any given voltage and [Na+] this block was identical in neutral and negatively charged membranes. These observations suggest that both the conduction pathway and the site(s) of Ca2+ block of the rat brain Na channel protein are electrostatically isolated from the negatively charged headgroups on the membrane lipids.     

Competitive binding interaction between Zn2+ and saxitoxin in cardiac Na+ channels. Evidence for a sulfhydryl group in the Zn2+/saxitoxin binding site.

Mammalian heart Na+ channels exhibit approximately 100-fold higher affinity for block by external Zn2+ than other Na+ channel subtypes. With batrachotoxin-modified Na+ channels from dog or calf heart, micromolar concentrations of external Zn2+ result in a flickering block to a substate level with a conductance of approximately 12% of the open channel at -50 mV. We examined the hypothesis that, in this blocking mode, Zn2+ binds to a subsite of the saxitoxin (STX) binding site of heart Na+ channels by single-channel analysis of the interaction between Zn2+ and STX and also by chemical modification experiments on single heart Na+ channels incorporated into planar lipid bilayers in the presence of batrachotoxin. We found that external Zn2+ relieved block by STX in a strictly competitive fashion. Kinetic analysis of this phenomenon was consistent with a scheme involving direct binding competition between Zn2+ and STX at a single site with intrinsic equilibrium dissociation constants of 30 nM for STX and 30 microM for Zn2+. Because high-affinity Zn2(+)-binding sites often include sulfhydryl groups as coordinating ligands of this metal ion, we tested the effect of a sulfhydryl-specific alkylating reagent, iodoacetamide (IAA), on Zn2+ and STX block. For six calf heart Na+ channels, we observed that exposure to 5 mM IAA completely abolished Zn2+ block and concomitantly modified STX binding with at least 20-fold reduction in affinity. These results lead us to propose a model in which Zn2+ binds to a subsite within or near the STX binding site of heart Na+ channels. This site is also presumed to contain one or more cysteine sulfhydryl groups.     

Functional consequences of lidocaine binding to slow-inactivated sodium channels

Na channels open upon depolarization but then enter inactivated states from which they cannot readily reopen. After brief depolarizations, native channels enter a fast-inactivated state from which recovery at hyperpolarized potentials is rapid (< 20 ms). Prolonged depolarization induces a slow-inactivated state that requires much longer periods for recovery (> 1 s). The slow-inactivated state therefore assumes particular importance in pathological conditions, such as ischemia, in which tissues are depolarized for prolonged periods. While use- dependent block of Na channels by local anesthetics has been explained on the basis of delayed recovery of fast-inactivated Na channels, the potential contribution of slow-inactivated channels has been ignored. The principal (alpha) subunits from skeletal muscle or brain Na channels display anomalous gating behavior when expressed in Xenopus oocytes, with a high percentage entering slow-inactivated states after brief depolarizations. This enhanced slow inactivation is eliminated by coexpressing the alpha subunit with the subsidiary beta 1 subunit. We compared the lidocaine sensitivity of alpha subunits expressed in the presence and absence of the beta 1 subunit to determine the relative contributions of fast-inactivated and slow-inactivated channel block. Coexpression of beta 1 inhibited the use-dependent accumulation of lidocaine block during repetitive (1-Hz) depolarizations from -100 to - 20 mV. Therefore, the time required for recovery from inactivated channel block was measured at -100 mV. Fast-inactivated (alpha + beta 1) channels were mostly unblocked within 1 s of repolarization; however, slow-inactivated (alpha alone) channels remained blocked for much longer repriming intervals (> 5 s). The affinity of the slow- inactivated state for lidocaine was estimated to be 15-25 microM, versus 24 microM for the fast-inactivated state. We conclude that slow- inactivated Na channels are blocked by lidocaine with an affinity comparable to that of fast-inactivated channels. A prominent functional consequence is potentiation of use-dependent block through a delay in repriming of lidocaine-bound slow-inactivated channels.