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Female↗
Biomedical subjects
S Spires
Publications and source records attributed to S Spires.
Voltage-independent gating transitions in squid axon potassium channels.
We have investigated the actions of internal and external Zn2+ on squid axon K channel ionic and gating currents. As has been noted previously, application of Zn2+ to either membrane surface substantially slowed the activation of these channels with little or no change in deactivation. Internal Zn2+ (near 200-300 nM) slowed channel activation by up to sixfold over the range of membrane voltages from -30 to +50 mV. External Zn2+ (10 mM) produced an approximate twofold slowing of activation from -40 to +40 mV. We found that the changes in ionic current activation kinetics were accompanied by less than a twofold slowing of channel-gating currents in a narrow range of potentials near -30 mV. There was, at most, only a few percent reduction of charge movement associated with Zn2+ application. We conclude that these ions interact with channel components involved in weakly voltage-dependent conformational changes. Although there are some differences in detail, the general similarity of the actions of both internal and external Zn2+ on channel function suggests that the modified channel-gating step involves amino acids accessible to both the internal and external membrane surface.
Animals↗
Modulation of potassium channel gating by external divalent cations.
We have examined the actions of Zn2+ ions on Shaker K channels. We found that low (100 microM) concentrations of Zn2+ produced a substantial (approximately three-fold) slowing of the kinetics of macroscopic activation and inactivation. Channel deactivation was much less affected. These results were obtained in the presence of 5 mM Mg2+ and 4 mM Ca2+ in the external solution and so are unlikely to be due to modification of membrane surface charges. Furthermore, the action of 100 microM Zn2+ on activation was equivalent to a 70-mV reduction of a negative surface potential whereas the effects on deactivation would require a 15-mV increase in surface potential. External H+ ions reduced the Zn-induced slowing of macroscopic activation with an apparent pK of 7.3. Treatment of Shaker K channels with the amino group reagent, trinitrobenzene sulfonic acid (TNBS), substantially reduced the effects of Zn2+. All these results are qualitatively similar to the actions of Zn2+ on squid K channels, indicating that the binding site may be a common motif in potassium channels. Studies of single Shaker channel properties showed that Zn2+ ions had little or no effect on the open channel current level or on the open channel lifetime. Rather, Zn2+ substantially delayed the time to first channel opening. Thus, K channels appear to contain a site to which divalent cations bind and in so doing act to slow one or more of the rate constants controlling transitions among closed conformational states of the channel.
Animals↗
Potassium channels in squid neuron cell bodies: comparison to axonal channels.
The squid giant axon is formed from the fusion of many axons whose cell bodies are located in the giant fiber lobe (GFL) of the stellate ganglion. We measured macroscopic potassium channel currents in both squid giant axons and in the cell bodies. These currents appear similar in many ways, but were differently affected by the amino group modifying reagent, trinitrobenzene sulfonic acid (TNBS). TNBS increased the steady-state amplitude of the axonal currents but decreased the somal currents. We also studied single K channel currents in the giant fiber lobe cells, for comparison with single K channel currents measured previously in the axons. We found two common classes of K channels, distinguished by their conductances (11 and 25 pS) and kinetic properties. The two channel types resemble the 10 and 20 pS channels that have been described in squid giant axons. These results suggest that the cell bodies and axons express fundamentally similar K channel types. The different effects of amino group modification on macroscopic K currents in the two regions may arise from small (perhaps posttranslational) modifications of the core proteins.
Amines↗
Na+ channels in cardiac and neuronal cells derived from a mouse embryonal carcinoma cell line.
1. Cells from a pluripotent murine embryonal carcinoma cell line (P19) were differentiated in vitro into cells with neurone- and cardiac-like phenotypes. Cells treated with 0.5 microM retinoic acid developed into neurone-like cells possessing extensive neurites. Dimethyl sulphoxide treatment (0.5%) produced large, spontaneously contracting cell aggregates with many properties of cardiac cells. 2. The neurone- and cardiac-like cells contained voltage-sensitive Na+ channels with properties similar to those of native neuronal and cardiac cells. 3. We used whole-cell patch clamp techniques to measure inward currents from the neurone- and cardiac-like cells. Undifferentiated (untreated) cells had only small inward currents (peak of -0.15 nA in 150 mM external Na+). The peak inward current in the neurone-like and cardiac-like cells was -1.2 nA (in 154 mM external Na+) and -2.8 nA (in only 46 mM Na+), respectively. These large currents were absent when the external solution contained no Na+. 4. Tetrodotoxin (TTX) blocked the Na+ currents in the neurone- and cardiac-like cells in a dose-dependent manner. The Kd for TTX block of the Na+ current in the neurone-like cells was 6.7 nM. The Na+ current in the cardiac-like cells was much more resistant to TTX; the half-blocking concentration was two orders of magnitude higher, 710 nM. 5. The kinetic properties of the Na+ channel currents in the neurone- and cardiac-like cells were similar but developed over somewhat different voltage ranges. The voltage sensitivity of activation was similar in both cell types but the activation mid-point voltage was different: -12 mV in the neuronal cells and -34 mV for cardiac cells. Inactivation of the neuronal Na+ channels had a mid-point near -47 mV and was more sensitive to the membrane voltage than inactivation of the cardiac channels. The mid-point of inactivation for the cardiac Na+ channels was -80 mV.
Animals↗
Chemical properties of the divalent cation binding site on potassium channels.
The actions of divalent cations on voltage-gated ion channels suggest that these cations bind to specific sites and directly influence gating kinetics. We have examined some chemical properties of the external divalent cation binding sites on neuronal potassium channels. Patch clamp techniques were used to measure the electrophysiological properties of these channels and Zn ions were used to probe the divalent cation binding site. The channel activation kinetics were greatly (three- to fourfold) slowed by low (2-5 mM) concentrations of Zn; deactivation kinetics were only slightly affected. These effects of Zn were inhibited by low solution pH in a manner consistent with competition between Zn and H ions for a single site. The apparent inhibitory pK for this site was near 7.2. Treatment of the neurons with specific amino acid reagents implicated amino, but no histidyl or sulfhydryl, residues in divalent cation binding.
Animals↗
Block of T-type Ca channels in guinea pig atrial cells by antiarrhythmic agents and Ca channel antagonists.
Myocardial cells have two types of Ca channels commonly called T-type and L-type. Whole cell Ca channel currents in guinea pig atrial myocytes can be separated and quantitated by analyzing channel closing kinetics after a brief depolarization (tail current analysis). L-type Ca channels deactivate rapidly when the membrane is repolarized and T-type Ca channels deactivate relatively slowly. Ca channel block by the therapeutically useful Ca channel antagonists is voltage dependent, so it is desirable to study block of both channel types over an extended voltage range. Tail current analysis allows this and was used to study block of both types of Ca channels under identical conditions. Amiodarone, bepridil, and cinnarizine block T-type Ca channels more potently than L-type Ca channels when binding equilibrates at normal diastolic potentials (approximately -90 mV). None of these drugs is a selective blocker of T-type Ca channels because block of L-type Ca channels is enhanced when cells are almost completely depolarized. Although weak block of T-type Ca channels by 1,4-dihydropyridines has usually been reported, we found that felodipine blocks these channels with high affinity. When most T-type Ca channels are inactivated, the apparent dissociation constant (KI) is 13 nM. Felodipine also blocks T-type Ca channels in GH3 cells (a cell line derived from rat anterior pituitary), but KI = 700 nM. Thus, T-type Ca channels in different cell types are pharmacologically distinct. Felodipine can block L-type Ca channels in atrial cells more potently than T-type Ca channels, but block of L-type Ca channels is potent only at depolarized potentials; block of both channel types is comparable at normal diastolic membrane potentials. Felodipine and the 1,4-dihydropyridines isradipine and (-)-202-791 are approximately equipotent at blocking T-type Ca channels, but differ substantially in potency for block of L-type Ca channels. Block of T-type Ca channels may account for some of the pharmacological effects of 1,4-dihydropyridines and for the antiarrhythmic activity of amiodarone and bepridil.
Animals↗
Modification of potassium channel kinetics by amino group reagents.
We have examined the actions of several amino group reagents on delayed rectifier potassium channels in squid giant axons. Three general classes of reagents were used: (1) those that preserved the positive charge of amino groups; (2) those that neutralize the charge; and (3) those that replace the positive with a negative charge. All three types of reagents produced qualitatively similar effects on K channel properties. Trinitrobenzene sulfonic acid (TNBS) neutralizes the peptide terminal amino groups and the epsilon-amino group of lysine groups. TNBS (a) slowed the kinetics of macroscopic ionic currents; (b) increased the size of ionic currents at large positive voltages; (c) shifted the voltage-dependent probability of channel opening to more positive potentials but had no effect on the voltage sensitivity; and (d) altered several properties of K channel gating currents. The actions of TNBS on gating currents suggest the presence of multiple gating current components. These effects are not all coupled, suggesting that several amino groups on the external surface of K channels are important for channel gating. A simple kinetic model that considers the channel to be composed of independent heterologous subunits is consistent with most of the modifications produced by amino group reagents.
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
Modification of potassium channel kinetics by histidine-specific reagents.
We have examined the actions of histidine-specific reagents on potassium channels in squid giant axons. External application of 20-500 microM diethylpyrocarbonate (DEP) slowed the opening of potassium channels with little or no effect on closing rates. Sodium channels were not affected by these low external concentrations of DEP. Internal application of up to 2 mM DEP had no effect on potassium channel kinetics. Steady-state potassium channel currents were reduced in an apparently voltage-dependent manner by external treatment with this reagent. The shape of the instantaneous current-voltage relation was not altered. The voltage-dependent probability of channel opening was shifted toward more positive membrane potentials, thus accounting for the apparent voltage-dependent reduction of steady-state current. Histidine-specific photo-oxidation catalyzed by rose bengal produced alterations in potassium channel properties similar to those observed with DEP. The rate of action of DEP was consistent with a single kinetic class of histidine residues. In contrast to the effects on ionic currents, potassium channel gating currents were not modified by treatment with DEP. These results suggest the existence of a histidyl group (or groups) on the external surface of potassium channels important for a weakly voltage-dependent conformational transition. These effects can be reproduced by a simple kinetic model of potassium channels.
Animals↗
Pharmacological and kinetic analysis of K channel gating currents.
We have measured gating currents from the squid giant axon using solutions that preserve functional K channels and with experimental conditions that minimize Na channel contributions to these currents. Two pharmacological agents were used to identify a component of gating current that is associated with K channels. Low concentrations of internal Zn2+ that considerably slow K channel ionic currents with no effect on Na channel currents altered the component of gating current associated with K channels. At low concentrations (10-50 microM) the small, organic, dipolar molecule phloretin has several reported specific effects on K channels: it reduces K channel conductance, shifts the relationship between channel conductance and membrane voltage (Vm) to more positive potentials, and reduces the voltage dependence of the conductance-Vm relation. The K channel gating charge movements were altered in an analogous manner by 10 microM phloretin. We also measured the dominant time constants of the K channel ionic and gating currents. These time constants were similar over part of the accessible voltage range, but at potentials between -40 and 0 mV the gating current time constants were two to three times faster than the corresponding ionic current values. These features of K channel function can be reproduced by a simple kinetic model in which the channel is considered to consist of two, two-state, nonidentical subunits.
Animals↗