Cesium effects on i(f) and i(K) in rabbit sinoatrial node myocytes: implications for SA node automaticity

Cesium blocks the hyperpolarization-activated current i(f) but blocks neither the delayed-rectifier current i(K) nor the sinoatrial (SA) node discharge. It has been proposed that the failure of Cs+ to block SA discharge is either an incomplete block or a negative shift of i(f). However, an alternative possibility is that i(K) (rather than i(f)) has a predominant role in the SA-pacemaker potential. To investigate this point, the effects of Cs+ on both i(f) and i(K) in the pacemaker range of potentials were studied in the same single SA node cell at the same time by means of the perforated patch-clamp technique. Hyperpolarizing steps from a holding potential (Vh) of -35 mV into and past the pacemaker-potential range resulted in a progressively larger i(f) associated with an increasing slope conductance. Cs+ (2 mM) reversibly blocked both i(f) and the slope conductance increase, suggesting that the current activated was indeed predominantly i(f). Subsequently, hyperpolarizing steps to -50, -60, and -70 mV were applied in the absence (to activate only i(f)) and in the presence of a prior depolarizing step to +10 mV (to activate i(K) as well, as the action potential normally does). Cs+ almost abolished i(f) but only slightly decreased i(K). It is concluded that the failure of Cs+ to block the SA- node spontaneous discharge is not due to a shift of i(f) out of the pacemaker range (due to run-down) or an incomplete block of i(f). Instead, the resistance of i(K) to block by Cs+ is consistent with a predominant role of i(K) for the discharge of the SA node, although i(f) can contribute under normal or special circumstances. The reduction of i(K) by Cs+ raises the question whether the Cs+ slows the SA-node discharge not only by suppressing I(f), but also by reducing i(K).     

Ion channel and exchange currents in single myocytes isolated from the rabbit atrioventricular node

OBJECTIVE: To review findings from the authors' laboratory in studies of electrophysiological properties of single rod- and spindle-shaped myocytes from rabbit atrioventricular node (AVN). DESIGN: Single cells were isolated from the AVN of the rabbit heart with the use of enzymatic and mechanical dispersion. For recording, cells were superfused with a Tyrode's solution at 33 to 37 degrees C, and recordings were made with microelectrodes or patch pipettes under 'current' or voltage' clamp conditions. Results are expressed as mean +/- SEM. RESULTS: AVN cells had a mean membrane capacitance of 40 +/- 3.9 pF and membrane resistance of 565 +/- 167 M omega (n = 9). Spontaneously active cells exhibited pacemaker activity showing a clear diastolic depolarization and overshooting action potential (AP) with a relatively slow upstroke velocity (7.4 +/- 0.9 V/s, n = 6) and a maximum diastolic potential of -70.5 +/- 2.9 mV. Under voltage clamp conditions, depolarizing pulses from 40 mV elicited L-type calcium currents sensitive to inhibition by nifedipine and managanese or cadmium ions, which could also block spontaneous APs. Depolarizing pulses also activated delayed rectifier potassium current (IK). IK showed rapid activation, and IK 'tails' in AVN cells were blocked by 5 microM E4031, consistent with the rapidly activating subtype of IK (IKr). IK was similar in AVN and ventricular myocytes, except for the time-course of deactivation, which was faster in AVN cells. In 80% to 90% of cells, hyperpolarizing voltage steps activated a small time-independent current. Ten per cent to 20% of cells showed the hyperpolarization-activated current (I(f)), but I(f) amplitude was only significant at potentials more negative than the pacemaker potential. AVN cells showed an apparent absence of inwardly rectifying potassium current. CONCLUSIONS: The high membrane resistance of AVN cells suggests that only small changes in ionic currents could significantly affect membrane potential. L-type calcium current is important in generating the AP upstroke, and IKr may play a role in both AP repolarization and diastolic depolarization. The ionic basis underlying spontaneous activity is not yet clear, but in some cells I(f) is not required because cells without I(f) can generate spontaneous APs.     

Modulation of C-type inactivation by K+ at the potassium channel selectivity filter

With prolonged or repetitive activation, voltage-gated K+ channels undergo a slow (C-type) inactivation mechanism, which decreases current flow through the channel. Previous observations suggest that C-type inactivation results from a localized constriction in the outer mouth of the channel pore and that the rate of inactivation is controlled by the-rate at which K+ leaves an unidentified binding site in the pore. We have functionally identified two K+ binding sites in the conduction pathway of a chimeric K+ channel that conducts Na+ in the absence of K+. One site has a high affinity for K+ and contributes to the selectivity filter mechanism for K+ over Na+. Another site, external to the high-affinity site, has a lower affinity for K+ and is not involved in channel selectivity. Binding of K+ to the high-affinity binding site slowed inactivation. Binding of cations to the external low-affinity site did not slow inactivation directly but could slow it indirectly, apparently by trapping K+ at the high-affinity site. These data support a model whereby C-type inactivation involves a constriction at the selectivity filter, and the constriction cannot proceed when the selectivity filter is occupied by K+.     

The structure of the potassium channel: molecular basis of K+ conduction and selectivity

The potassium channel from Streptomyces lividans is an integral membrane protein with sequence similarity to all known K+ channels, particularly in the pore region. X-ray analysis with data to 3.2 angstroms reveals that four identical subunits create an inverted teepee, or cone, cradling the selectivity filter of the pore in its outer end. The narrow selectivity filter is only 12 angstroms long, whereas the remainder of the pore is wider and lined with hydrophobic amino acids. A large water-filled cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore at the center of the bilayer. Main chain carbonyl oxygen atoms from the K+ channel signature sequence line the selectivity filter, which is held open by structural constraints to coordinate K+ ions but not smaller Na+ ions. The selectivity filter contains two K+ ions about 7.5 angstroms apart. This configuration promotes ion conduction by exploiting electrostatic repulsive forces to overcome attractive forces between K+ ions and the selectivity filter. The architecture of the pore establishes the physical principles underlying selective K+ conduction.     

Structural model of the outer vestibule and selectivity filter of the Shaker voltage-gated K+ channel

A new generation of structural models were developed of the outer vestibule and ion-selective portion of the voltage-gated Shaker K+ channel. Some features of these models are similar to those that we have developed previously [Durrel S. R. and Guy H. R. (1992) Biophys. J. 62, 238-250; Guy H. R. (1990) In Monovalent Cations in Biological Systems (Pasternak C. A., Ed.), pp. 31-58, CRC Press, Boca Raton, FL; Guy H. R. and Durell S. R. (1994) In Molecular Evolution of Physiological processes (Fambrough D., Ed.), pp. 197-212, The Rockefeller University Press, NY; Guy H. R. and Durell S. R. (1995) In Ion Channels and Genetic Diseases (Dawson D., Ed.), pp. 1-16, The Rockefeller University Press, NY] and other features were modified to make the models more consistent with recent experimental findings. The first part of the P segment is postulated, as always, to form a short alpha helix that spans only the outer portion of the membrane. The helix is tilted so that its C-terminal is nearer the pore than its N-terminal. The latter part of the P segment, P2, is postulated to have a relatively elongated conformation that is positioned approximately parallel to the axis of the pore. Four of the P2 segments assemble to form an ion-selective region that has two narrow regions; one formed by the Y445 side-chains at the outer entrance of the pore and one formed by the backbone of the T442 residues near the innermost part of the P segments. The S6 segment is postulated to form two alpha helices. The first S6 helix packs next to the P segments in our models. The NMR structures of two scorpion toxins, charybdotoxin and agitoxin 2, have been docked into the models of the outer vestibules. The shape of the outer vestibule has been modeled so that specific toxin-channel residue-residue interactions correspond to those that have been identified experimentally.     

Potentiometric study of resting potential, contributing K+ channels and the onset of Na+ channel excitability in embryonic rat cortical cells

Resting membrane potential (RMP), K+ channel contribution to RMP and the development of excitability were investigated in the entire population of acutely dissociated embryonic (E) rat cortical cells over E11-22 using a voltage-sensitive fluorescent indicator dye and flow cytometry. During the period of intense proliferation (E11-13), two cell subpopulations with distinct estimated RMPs were recorded: one polarized at approximately -70 mV and the other relatively less-polarized at approximately -40 mV. Ca2+o was critical in sustaining the RMP of the majority of less-polarized cells, while the well-polarized cells were characterized by membrane potentials exhibiting a approximately Nernstian relationship between RMP and [K+]o. Analysis of these two subpopulations revealed that > 80% of less-polarized cells were proliferative, while > 90% of well-polarized cells were postmitotic. Throughout embryonic development, the disappearance of Ca2+o-sensitive, less-polarized cells correlated with the disappearance of the proliferating population, while the appearance of the K+o-sensitive, well-polarized population correlated with the appearance of terminally postmitotic neurons, immuno-identified as BrdU-, tetanus toxin+ cells. Differentiating neurons were estimated to contain increased K+i relative to less-polarized cells, coinciding with the developmental expression of Cs+/Ba2+-sensitive and Ca2+-dependent K+ channels. Both K+ channels contributed to the RMP of well-polarized cells, which became more negative toward the end of neurogenesis. Depolarizing effects of veratridine, first observed at E11, progressively changed from Ca2+o-dependent and tetrodotoxin-insensitive to Na+o-dependent and tetrodotoxin-sensitive response by E18. The results reveal a dynamic development of RMP, contributing K+ channels and voltage-dependent Na+ channels in the developing cortex as it transforms from proliferative to primarily differentiating tissue.     

Spatial localization of the K+ channel selectivity filter by mutant cycle-based structure analysis

The structurally well-characterized scorpion toxin Agitoxin2 inhibits ion permeation through Shaker K+ channels by binding to the external pore entryway. Scanning mutagenesis identified a set of inhibitor residues critical for making energetic contacts with the channel. Using thermodynamic mutant cycle analysis, we have mapped channel residues relative to the known inhibitor structure. This study constrains the position of multiple channel residues within the pore-forming loops; in one stretch, we have been able to map five out of seven contiguous residues to the inhibitor interaction surface, including those involved in ion selectivity. One interaction in particular, that of K27M on the inhibitor with Y445F on the channel, is unique in that it depends on the K+ ion concentration. These results reveal a shallow vestibule formed by the pore loops at the K+ channel entryway. The selectivity filter is located at the center of the vestibule close to (approximately 5 A) the extracellular solution.     

Overexpression of the Na+,K+-ATPase alpha1 subunit increases Na+,K+-ATPase function in A549 cells

Ron (the receptor for Macrophage Stimulating Protein) has never been implicated before in human malignancies or in cell transformation. In this report we show that Ron can acquire oncogenic potential by means of two amino acid substitutions-D1232V and M1254T-affecting highly conserved residues in the tyrosine kinase domain. The same mutations in Kit and Ret have been found associated with two human malignancies, mastocytosis and Multiple Endocrine Neoplasia type 2B (MEN2B), respectively. Both mutations caused Ron-mediated transformation of 3T3 fibroblasts and tumour formation in nude mice. Moreover, cells transformed by the oncogenic Ron mutants displayed high metastatic potential. The Ron mutant receptors were constitutively active and the catalytic efficiency of the mutated kinase was higher than that of wild-type Ron. Oncogenic Ron mutants enhanced activation of the Ras/MAPK cascade with respect to wild type Ron, without affecting the JNK/SAPK pathway. Expression of Ron mutants in 3T3 fibroblasts led to different patterns of tyrosine-phos-phorylated proteins. These data show that point mutations altering catalytic properties and possibly substrate specificity of the Ron kinase may force cells toward tumorigenesis and metastasis.     

Cooperative binding of the organophosphate paraoxon to the (Na+ + K+)-ATPase

Paraoxon, the main active metabolite of the organophosphorus insecticide parathion, exerted a dose-dependent inhibitory effect on the activity of pig kidney (Na+ + K+)-ATPase contained in microsomal fraction and purified from it. Substrate kinetics studies revealed the existence of two active sites with high and low affinity to ATP. The Dixon analysis of the mode of the inhibition indicated its noncompetitive character. The purified enzyme was more affected than enzyme contained in the microsomal fraction. The inhibition constant Ki ranged from 73 to 245 microM depending on the type of preparation. The Hill coefficient (n) fulfilled the relationship 1 < n < 3. These properties of the interaction suggest the cooperative binding of paraoxon to the enzyme. An indirect mechanism of the interaction was proposed: paraoxon could inhibit the activity of the (Na+ + K+)-ATPase by excluding the enzyme protein from its normal lipid milieu.     

Nedd4 mediates control of an epithelial Na+ channel in salivary duct cells by cytosolic Na+

Epithelial Na+ channels are expressed widely in absorptive epithelia such as the renal collecting duct and the colon and play a critical role in fluid and electrolyte homeostasis. Recent studies have shown that these channels interact via PY motifs in the C terminals of their alpha, beta, and gamma subunits with the WW domains of the ubiquitin-protein ligase Nedd4. Mutation or deletion of these PY motifs (as occurs, for example, in the heritable form of hypertension known as Liddle's syndrome) leads to increased Na+ channel activity. Thus, binding of Nedd4 by the PY motifs would appear to be part of a physiological control system for down-regulation of Na+ channel activity. The nature of this control system is, however, unknown. In the present paper, we show that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. We further show that Nedd4 operates downstream of Go in this feedback pathway. We find, however, that Nedd4 is not involved in the feedback control of Na+ channels by intracellular anions. Finally, we show that Nedd4 has no influence on Na+ channel activity when the Na+ and anion feedback systems are inactive. We conclude that Nedd4 normally mediates feedback control of epithelial Na+ channels by intracellular Na+, and we suggest that the increased Na+ channel activity observed in Liddle's syndrome is attributable to the loss of this regulatory feedback system.     

Dual effect of nitric oxide on the hyperpolarization-activated inward current (I(f)) in sino-atrial node cells of the rabbit

Using the whole-cell voltage-clamp technique, we have investigated the effect of nitric oxide (NO) donor (sodium nitroprusside, SNP) on hyperpolarization-activated inward current, I(f), in isolated rabbit sinoatrial node (SAN) cells. I(f) in the basal state increased when NO was applied but decreased when I(f) was pre-stimulated by isoproterenol (ISO) or by adding cAMP to the pipette solution. Both the stimulatory and the inhibitory effects of NO were abolished by guanylyl cyclase inhibitor, methylene blue (MB), suggesting that the effect of NO is mediated by cGMP. The inhibitory effect of NO was abolished when I(f) was pre-stimulated by 3-isobutyl-1-methylxanthine (IBMX), which is a phosphodiesterase (PDE) inhibitor, or by adding 8Br-cAMP (which is resistant to PDE) to the pipette solution. An analogue of cGMP, 8Br-cGMP, which is a potent stimulator of cGMP-dependent protein kinase (PKG) but has little effect on PDE, did not inhibit I(f) when I(f) was pre-stimulated by ISO. In its basal state, I(f) was still increased by 8Br-cGMP, and this effect was not prevented by the pretreatment with H-7, PKG inhibitor. The effect of acetylcholine (ACh) was not identical to that of NO: I(f) decreased when pre-stimulated not only by ISO, but also by IBMX. The above results suggest that via cGMP, NO exerts a dual effect on I(f): the inhibitory effect is mediated by cGMP-stimulated PDE, and the stimulatory effect may be attributable to direct binding of cGMP to I(f) channels.     

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.     

Fatty acids suppress voltage-gated Na+ currents in HEK293t cells transfected with the alpha-subunit of the human cardiac Na+ channel

Studies have shown that fish oils, containing n-3 fatty acids, have protective effects against ischemia-induced, fatal cardiac arrhythmias in animals and perhaps in humans. In this study we used the whole-cell voltage-clamp technique to assess the effects of dietary, free long-chain fatty acids on the Na+ current (INa,alpha) in human embryonic kidney (HEK293t) cells transfected with the alpha-subunit of the human cardiac Na+ channel (hH1alpha). Extracellular application of 0.01 to 30 microM eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced INa,alpha with an IC50 of 0.51 +/- 0.06 microM. The EPA-induced suppression of INa,alpha was concentration- and voltage-dependent. EPA at 5 microM significantly shifted the steady-state inactivation relationship by -27.8 +/- 1.2 mV (n = 6, P < 0.0001) at the V1/2 point. In addition, EPA blocked INa,alpha with a higher "binding affinity" to hH1alpha channels in the inactivated state than in the resting state. The transition from the resting state to the inactivated state was markedly accelerated in the presence of 5 microM EPA. The time for 50% recovery from the inactivation state was significantly slower in the presence of 5 microM EPA, from 2.1 +/- 0.8 ms for control to 34.8 +/- 2.1 ms (n = 5, P < 0.001). The effects of EPA on INa,alpha were reversible. Furthermore, docosahexaenoic acid (C22:6n-3), alpha-linolenic acid (C18:3n-3), conjugated linoleic acid (C18:2n-7), and oleic acid (C18:1n-9) at 5 microM and all-trans-retinoic acid at 10 microM had similar effects on INa,alpha as EPA. Even 5 microM of stearic acid (C18:0) or palmitic acid (C16:0) also significantly inhibited INa, alpha. In contrast, 5 microM EPA ethyl ester did not alter INa,alpha (8 +/- 4%, n = 8, P > 0.05). The present data demonstrate that free fatty acids suppress INa,alpha with high "binding affinity" to hH1alpha channels in the inactivated state and prolong the duration of recovery from inactivation.     

Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel (hslo)

Large conductance calcium- and voltage-sensitive K+ (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca2+ favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca2+ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4-5 elementary charges.     

Subcellular distribution of ankyrin in developing rabbit heart--relationship to the Na+-Ca2+ exchanger

Ankyrins are a multigene family of proteins that function as adapters between the cytoskeleton and trans-membrane proteins, such as ion channels. Previous studies have shown the linkage between ankyrin and ionic transport proteins such as Na+-K+ ATPase, voltage-dependent Na+ channels and Ca2+ channels. In the present study, we have investigated the subcellular distribution of ankyrin and its relationship to the Na+-Ca2+ exchange protein in immature and adult rabbit ventricular myocytes. Isolated single cardiomyocytes from neonatal, juvenile and adult rabbit hearts were examined by immunofluorescence labeling techniques, using antibodies against ankyrin and the Na+-Ca2+ exchanger. We found that in neonatal rabbit cardiac myocytes, ankyrin labeling was mainly present at the Z disk, whereas the Na+-Ca2+ exchanger was only present on the peripheral sarcolemma. At 2 weeks of age, ankyrin labeling was still predominantly observed at the level of the Z disks as well as in the partially developed T-tubules. In the adult cells, however, ankyrin and the Na+-Ca2+ exchanger seem to be co-localized within T-tubules and at the costamere region of the peripheral sarcolemma. Immunogold labeling studies at the higher resolution electron microscopic level using cyrosection tissues of rabbit heart at different ages confirm these findings. These results indicate that the distribution pattern of ankyrin and the Na+-Ca2+ exchanger changes with development in rabbit ventricular myocytes.     

Electrophysiological effects of ketamine on Ca(2+)-activated K+ channel in single rabbit portal vein cell

The effects of ketamine on Ca(2+)-activated K+ channel currents were studied in dispersed single smooth muscle cells from rabbit portal vein using inside-out patch clamp technique. In a near physiological K+ and Ca2+ gradient, three populations of outward rectangular single currents were recorded in isolated cell membrane of rabbit portal vein at +60 mV membrane potential. These currents were judged as Ca(2+)-activated K+ channel currents since application of EGTA or Apamin in the internal solution inhibited these currents. Application of 10(-5)M or 10(-4)M ketamine inhibited the number of occurrences of channel opening and decreased open times, but did not reduce the amplitudes. When the 10(-3)M ketamine was applied, the Ca(2+)-activated K+ channel currents were abolished. We suggest that the depression of Ca(2+)-activated K+ channel currents may explain the continuous contraction observed in rabbit portal vein at a clinical concentration of ketamine from a point of electrophysiological K+ current study.