Increasing small conductance Ca2+-activated potassium channel activity reverses ischemia-induced impairment of long-term potentiation.

Global cerebral ischemia following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) causes injury to hippocampal CA1 pyramidal neurons and impairs cognition. Small conductance Ca(2+)-activated potassium channels type 2 (SK2), expressed in CA1 pyramidal neurons, have been implicated as potential protective targets. Here we showed that, in mice, hippocampal long-term potentiation (LTP) was impaired as early as 3 h after recovery from CA/CPR and LTP remained impaired for at least 30 days. Treatment with the SK2 channel agonist 1-Ethyl-2-benzimidazolinone (1-EBIO) at 30 min after CA provided sustained protection from plasticity deficits, with LTP being maintained at control levels at 30 days after recovery from CA/CPR. Minimal changes in glutamate release probability were observed at delayed times after CA/CPR, implicating post-synaptic mechanisms. Real-time quantitative reverse transcriptase-polymerase chain reaction indicated that CA/CPR did not cause a loss of N-methyl-D-aspartate (NMDA) receptor mRNA at 7 or 30 days after CA/CPR. Similarly, no change in synaptic NMDA receptor protein levels was observed at 7 or 30 days after CA/CPR. Further, patch-clamp experiments demonstrated no change in functional synaptic NMDA receptors at 7 or 30 days after CA/CPR. Electrophysiology recordings showed that synaptic SK channel activity was reduced for the duration of experiments performed (up to 30 days) and that, surprisingly, treatment with 1-EBIO did not prevent the CA/CPR-induced loss of synaptic SK channel function. We concluded that CA/CPR caused alterations in post-synaptic signaling that were prevented by treatment with the SK2 agonist 1-EBIO, indicating that activators of SK2 channels may be useful therapeutic agents to prevent ischemic injury and cognitive impairments.

Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy.

Expansion of a CTG trinucleotide repeat in the 3' UTR of the gene DMPK at the DM1 locus on chromosome 19 causes myotonic dystrophy, a dominantly inherited disease characterized by skeletal muscle dystrophy and myotonia, cataracts and cardiac conduction defects. Targeted deletion of Dm15, the mouse orthologue of human DMPK, produced mice with a mild myopathy and cardiac conduction abnormalities, but without other features of myotonic dystrophy, such as myotonia and cataracts. We, and others, have demonstrated that repeat expansion decreases expression of the adjacent gene SIX5 (refs 7,8), which encodes a homeodomain transcription factor. To determine whether SIX5 deficiency contributes to the myotonic dystrophy phenotype, we disrupted mouse Six5 by replacing the first exon with a beta-galactosidase reporter. Six5-mutant mice showed reporter expression in multiple tissues, including the developing lens. Homozygous mutant mice had no apparent abnormalities of skeletal muscle function, but developed lenticular opacities at a higher rate than controls. Our results suggest that SIX5 deficiency contributes to the cataract phenotype in myotonic dystrophy, and that myotonic dystrophy represents a multigenic disorder.

Small-conductance, calcium-activated potassium channels from mammalian brain.

Members of a previously unidentified family of potassium channel subunits were cloned from rat and human brain. The messenger RNAs encoding these subunits were widely expressed in brain with distinct yet overlapping patterns, as well as in several peripheral tissues. Expression of the messenger RNAs in Xenopus oocytes resulted in calcium-activated, voltage-independent potassium channels. The channels that formed from the various subunits displayed differential sensitivity to apamin and tubocurare. The distribution, function, and pharmacology of these channels are consistent with the SK class of small-conductance, calcium-activated potassium channels, which contribute to the afterhyperpolarization in central neurons and other cell types.

Respiration and parturition affected by conditional overexpression of the Ca2+-activated K+ channel subunit, SK3.

In excitable cells, small-conductance Ca2+-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor.

Persistent activation of min K channels by chemical cross-linking.

Expression of the structurally and functionally distinct min K channel in Xenopus oocytes results in voltage-dependent potassium currents that activate with a characteristic slow time course. Application of a membrane-impermeable chemical cross-linking agent to oocytes expressing min K decreased the time-dependent current, increased its rate of activation, and induced persistently activated inward and outward potassium currents. These effects required membrane depolarization, demonstrating use dependence. Persistently activated channels retained potassium selectivity and sensitivity to block by clofilium and barium. These results suggest that a major conformational change occurs during min K channel gating, which can be stabilized by chemical cross-linking, and are consistent with a model in which min K channels activate by voltage-dependent subunit aggregation.

Episodic ataxia results from voltage-dependent potassium channels with altered functions.

Episodic ataxia (EA) is an autosomal dominant human disorder that produces persistent myokymia and attacks of generalized ataxia. Recently, familial EA has been linked to the voltage-dependent delayed rectifier, Kv1.1, on chromosome 12. Six EA families have been identified that carry distinct Kv1.1 missense mutations; all individuals are heterozygous. Expression in Xenopus oocytes demonstrates that two of the EA subunits form homomeric channels with altered gating properties. V408A channels have voltage dependence similar to that of wild-type channels, but with faster kinetics and increased C-type inactivation, while the voltage dependence of F184C channels is shifted 20 mV positive. The other four EA subunits do not produce functional homomeric channels but reduce the potassium current when coassembled with wild-type subunits. The results suggest a cellular mechanism underlying EA in which the affected nerve cells cannot efficiently repolarize following an action potential because of altered delayed rectifier function.

Domains responsible for constitutive and Ca(2+)-dependent interactions between calmodulin and small conductance Ca(2+)-activated potassium channels.

Small conductance Ca(2+)-activated potassium channels (SK channels) are coassembled complexes of pore-forming SK alpha subunits and calmodulin. We proposed a model for channel activation in which Ca2+ binding to calmodulin induces conformational rearrangements in calmodulin and the alpha subunits that result in channel gating. We now report fluorescence measurements that indicate conformational changes in the alpha subunit after calmodulin binding and Ca2+ binding to the alpha subunit-calmodulin complex. Two-hybrid experiments showed that the Ca(2+)-independent interaction of calmodulin with the alpha subunits requires only the C-terminal domain of calmodulin and is mediated by two noncontiguous subregions; the ability of the E-F hands to bind Ca2+ is not required. Although SK alpha subunits lack a consensus calmodulin-binding motif, mutagenesis experiments identified two positively charged residues required for Ca(2+)-independent interactions with calmodulin. Electrophysiological recordings of SK2 channels in membrane patches from oocytes coexpressing mutant calmodulins revealed that channel gating is mediated by Ca2+ binding to the first and second E-F hand motifs in the N-terminal domain of calmodulin. Taken together, the results support a calmodulin- and Ca(2+)-calmodulin-dependent conformational change in the channel alpha subunits, in which different domains of calmodulin are responsible for Ca(2+)-dependent and Ca(2+)-independent interactions. In addition, calmodulin is associated with each alpha subunit and must bind at least one Ca2+ ion for channel gating. Based on these results, a state model for Ca2+ gating was developed that simulates alterations in SK channel Ca2+ sensitivity and cooperativity associated with mutations in CaM.

Structure and complex transcription pattern of the mouse SK1 K(Ca) channel gene, KCNN1.

Small conductance calcium-gated K(+) channels (SK channels) are encoded by the three SK genes, SK1, SK2, and SK3. These channels likely contribute to slow synaptic afterhyperpolarizations of apamin-sensitive and apamin-insensitive types. SK channels are also widely expressed outside the nervous system. The mouse SK1 gene comprises at least 12 exons extending across 19.8 kb of genomic DNA. This gene encodes a complex pattern of alternatively spliced SK1 transcripts widely expressed among mouse tissues. These transcripts exhibit at least four distinct 5'-nucleotide sequence variants encoding at least two N-terminal amino acid sequences. Optional inclusion of exons 7 and 9, together with two alternate splice donor sites in exon 8, yields transcripts encoding eight variant C-terminal amino acid sequences for SK1. These include an altered putative S6 transmembrane span, modification of the C-terminal cytoplasmic domain binding site for calmodulin, and generation of two alternate predicted binding sites for PDZ domain-containing proteins. 20 of the 32 predicted mouse SK1 transcripts are expressed in brain at levels sufficient to allow consistent detection, and encode 16 SK1 polypeptide variants. Only four of these 16 polypeptides preserve the ability to bind calmodulin in a Ca(2+)-independent manner. Mouse SK1 also exhibits novel, strain-specific, length polymorphism of a polyglutamate repeat in the N-terminal cytoplasmic domain. The evolutionary conservation of this complex transcription pattern suggests a possible role in the tuning of SK1 channel function.

Intrinsic optical and passive electrical properties of cut frog twitch fibers.

This article describes a new apparatus for making simultaneous optical measurements on single muscle fibers at three different wavelengths and two planes of linear polarization. There are two modes of operation: mode 1 measures the individual absorbances of light linearly polarized along and perpendicular to the fiber axis, and mode 2 measures retardation (or birefringence) and the average of the two absorbance components. Although some intact frog twitch fibers were studied, most experiments used cut fibers (Hille, B., and D. T. Campbell. 1976. Journal of General Physiology. 67:265-293) mounted in a double-Vaseline-gap chamber (Kovacs, L., E. Rios, and M. F. Schneider. 1983. Journal of Physiology. 343:161-196). The end-pool segments were usually exposed for 2 min to 0.01% saponin. This procedure, used in subsequent experiments to make the external membranes in the end pools permeable to Ca indicators (Maylie, J., M. Irving, N. L. Sizto, G. Boyarsky, and W. K. Chandler. 1987. Journal of General Physiology. 89:145-176; Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:41-143), was routinely employed so that all our cut fiber results would be comparable. A simple method, which does not require microelectrodes, allowed continual estimation of a fiber's membrane (rm) and internal longitudinal (ri) resistances as well as the external resistance (re) under the Vaseline seals. The values of rm and ri obtained from cut fibers with this method agree reasonably well with values obtained from intact fibers using microelectrode techniques. Optical measurements were made on resting and action potential-stimulated fibers. The intrinsic fiber absorbance, defined operationally as log10 of the ratio of incident light to transmitted light intensity, was similar in intact and cut preparations, as were the changes that accompanied stimulation. On the other hand, the resting birefringence and the peak of the active change in cut fibers were, respectively, only 0.8 and 0.7 times the corresponding values in intact fibers. Both the amplitude and the half-width of the active retardation signal increased considerably during the time course of cut fiber experiments; a twofold increase in 2 h was not unusual. Such changes are probably due to a progressive alteration in the internal state of the cut fibers.

Calcium signals recorded from cut frog twitch fibers containing tetramethylmurexide.

The Ca indicator tetramethylmurexide was introduced into cut fibers, mounted in a double-Vaseline-gap chamber, by diffusion from the end-pool solutions. The indicator diffused rapidly to the central region of a fiber where optical recording was done and, if removed, diffused away equally fast. The time course of concentration suggests that, on average, a fraction 0.27 of indicator was reversibly bound to myoplasmic constituents and the free diffusion constant was 1.75 x 10(-6) cm2/s at 18 degrees C. The shape of the resting absorbance spectrum suggests that a fraction 0.11-0.15 of tetramethylmurexide inside a fiber was complexed with Ca. After action potential stimulation, there was a rapid transient change in indicator absorbance followed by a maintained change of opposite sign. The wavelength dependence of both changes matched a cuvette Ca-difference spectrum. The amplitude of the early peak varied linearly with indicator concentration and corresponded to an average rise in free [Ca] of 17 microM. These rather diverse findings can be explained if the sarcoplasmic reticulum membranes are permeable to Ca-free indicator. Both Ca-free and Ca-complexed indicator inside the sarcoplasmic reticulum would appear to be bound by diffusion analysis and the Ca-complexed form would be detected by the resting absorbance spectrum. The transient change in indicator absorbance would be produced by myoplasmic Ca reacting with indicator molecules that freely diffuse in myoplasmic solution. The maintained signal, which reports Ca dissociating from indicator complexed at rest, would come from changes within the sarcoplasmic reticulum. A method, based on these ideas, is described for separating the two components of the tetramethylmurexide signal. The estimated myoplasmic free [Ca] transient has an average peak value of 26 microM at 18 degrees C. Its time course is similar to, but possibly faster than, that recorded with antipyrylazo III (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:83-143).

Comparison of arsenazo III optical signals in intact and cut frog twitch fibers.

The Ca indicator arsenazo III was introduced into cut frog twitch fibers by diffusion from end-pool segments rendered permeable by saponin. After 2-3 h, the arsenazo III concentration at the optical recording site in the center of a fiber reached two to three times that in the end-pool solutions. Thus, arsenazo III was bound to or taken up by intracellular constituents. The time course of indicator appearance was fitted by equations for diffusion plus linear reversible binding; on average, 0.73 of the indicator was bound and the free diffusion constant was 0.86 x 10(-6) cm2/s at 18 degrees C. When the indicator was removed from the end pools, it failed to diffuse away from the optical site as rapidly as it had diffused in. The wavelength dependence of resting arsenazo III absorbance was the same in cut fibers and injected intact fibers. After action potential stimulation, the active Ca and dichroic signals were similar in the two preparations, which indicates that arsenazo III undergoes the same changes in absorbance and orientation in both cut and intact fibers. Ca transients in freshly prepared cut fibers appeared to be similar to those in intact fibers. As a cut fiber experiment progressed, however, the Ca signal changed. With action potential stimulation, the half-width of the signal gradually increased, regardless of whether the indicator concentration was increasing or decreasing. This increase was usually not accompanied by any change in the amplitude of the Ca signal at a given indicator concentration or by any obvious deterioration in the electrical condition of the fiber. In voltage-clamp experiments near threshold, the relation between peak [Ca] and voltage usually became less steep with time and shifted to more negative potentials. All these changes were also observed in cut fibers containing antipyrylazo III (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:83-143). They are considered to represent a progressive change in the physiological state of a cut fiber during the time course of an experiment.

Calcium signals recorded from cut frog twitch fibers containing antipyrylazo III.

The Ca indicator antipyrylazo III was introduced into cut frog twitch fibers by diffusion (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:41-81). Like arsenazo III, antipyrylazo III was largely bound to or sequestered by intracellular constituents; on average, a fraction 0.68 was so immobilized. After action potential stimulation, there was an early change in absorbance, with a wavelength dependence that nearly matched a cuvette Ca-difference spectrum. As with arsenazo III, this signal became prolonged as experiments progressed. In a freshly prepared cut fiber containing 0.3 mM indicator, the absorbance change had an average half-width of 10 ms at 18 degrees C. The peak amplitude of this Ca signal depended on the indicator concentration in a roughly parabolic manner, which is consistent with a 1:2 stoichiometry for Ca:indicator complexation and, for indicator concentrations less than or equal to 0.4 mM, constant peak free [Ca]. If all the antipyrylazo III inside a fiber can react normally with Ca, peak free [Ca] is 3 microM at 18 degrees C. If only freely diffusible indicator can react, the estimate is 42 microM. The true amplitude probably lies somewhere in between. The time course of Ca binding to intracellular buffers and of Ca release from the sarcoplasmic reticulum is estimated from the 3- and 42-microM myoplasmic [Ca] transients. After action potential stimulation, the release waveform is rapid and brief; its latency after the surface action potential is 2-3 ms and its half-width is 2-4 ms. This requires rapid coupling between the action potential in the transverse tubular system and Ca release from the sarcoplasmic reticulum. The peak fractional occupancy calculated for Ca-regulatory sites on troponin is 0.46 for the 3-microM transient and 0.93 for the 42-microM transient. During a 100-ms tetanus at 100 Hz, the corresponding fractional occupancies are 0.56 and 0.94. The low value of occupancy associated with the low-amplitude [Ca] calibration seems inconsistent with a brief tetanus being able to produce near-maximal activation (Blinks, J. R., R. Rudel, and S. R. Taylor. 1978. Journal of Physiology. 277:291-323; Lopez J. R., L. A. Wanck, and S. R. Taylor. 1981. Science. 214:47-82).

Gating of recombinant small-conductance Ca-activated K+ channels by calcium.

Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 micro M Ca2+ and was maximal above 2 micro M Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was approximately 0.6 in 1 micro M free Ca2+. A lower open probability of approximately 0.05 in 1 micro M Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches.

Simultaneous monitoring of changes in magnesium and calcium concentrations in frog cut twitch fibers containing antipyrylazo III.

Antipyrylazo III was introduced into frog cut twitch fibers (17-19 degrees C) by diffusion. After action potential stimulation, the change in indicator absorbance could be resolved into two components that had different time courses and wavelength dependences. The first component was early and transient and due to an increase in myoplasmic free [Ca] (Maylie, J., M. Irving, N.L. Sizto, and W.K. Chandler, 1987, Journal of General Physiology, 89:83-143). The second component, usually measured at 590 nm (near the isosbestic wavelength for Ca), developed later than the Ca transient and returned towards baseline about 100 times more slowly. Although the wavelength dependence of this component is consistent with an increase in either free [Mg] or pH, its time course is clearly different from that of the signals obtained with the pH indicators phenol red and 4',5'-dimethyl-5-(and -6-) carboxyfluorescein, suggesting that it is mainly due to an increase in free [Mg]. After a single action potential in freshly prepared cut fibers that contained 0.3 mM antipyrylazo III, the mean peak amplitude of delta A (590) would correspond to an increase in free [Mg] of 47 microM if all the signal were due to a change in [Mg] and all the intracellular indicator reacted with Mg as in cuvette calibrations. With either repetitive action potential stimulation or voltage-clamp depolarization, the delta A (590) signal continued to develop throughout the period when free [Ca] was elevated and then recovered to within 40-90% of the prestimulus baseline with an average rate constant between 0.5 and 1.0 s-1. With prolonged voltage-clamp depolarization, both the amplitude and rate of development of the delta A(590) signal increased with the amplitude of the depolarization and appeared to saturate at levels corresponding to an increase in free [Mg] of 0.8-1.4 mM and a maximum rate constant of 3-4 s-1, respectively. These results are consistent with the idea that the delta A(590) signal is primarily due to changes in myoplasmic free [Mg] produced by a change in the Mg occupancy of the Ca,Mg sites on parvalbumin that results from the Ca transient.

Hypotonic-induced stretch counteracts the efficacy of the class III antiarrhythmic agent E-4031 in guinea pig myocytes.

OBJECTIVES: The aim was to determine the effect and mechanisms by which myocyte stretch interacts with the prolongation of action potential duration (APD) by the class III antiarrhythmic agent E-4031. METHODS: Action potentials and whole-cell currents were measured in isolated guinea pig ventricular myocytes with a patch clamp procedure during perfusion of normotonic, normotonic with addition of E-4031, and hypotonic plus E-4031 solutions. RESULTS: Cell swelling leading to membrane stretch of myocytes in the whole-cell recording configuration occurred with hypotonic solution perfusion. APD, prolonged by E-4031, was reduced to less than control value with hypotonic-induced stretch. Evaluation of whole-cell currents after hypotonic-induced stretch revealed no significant changes in the L-type Ca2+ current, inward rectifier K+ current or the rapid component of the delayed rectifier K+ current. The slow component of the delayed rectifier K+ current (IKs) was upregulated and a stretch-induced CI- current was activated in hypotonic solutions. The hypotonic-induced modulation of these currents was not effected by protein kinase A or C inhibition. CONCLUSIONS: Hypotonic-induced stretch shortens APD and counteracts the effects of E-4031. This APD shortening is secondary to upregulation of IKs and activation of a stretch-induced Cl- current.

Characterization of three episodic ataxia mutations in the human Kv1.1 potassium channel.

Episodic ataxia (EA) is a rare inherited neurological disorder due to mutation in the voltage-dependent Kv1.1 potassium channel. In nine unrelated families, a different missense point mutation at highly conserved positions has been reported. We have previously characterized six of the EA mutants. In this study, three recently identified mutations were introduced into the human Kv1.1 cDNA and expressed in Xenopus oocytes. Compared to wild type, T226A and T226M reduced the current amplitude by > 95%, shifted the voltage dependence by 15 mV, and slowed activation and deactivation kinetics. Currents from G311S were approximately 25% of wild type, less steeply voltage-dependent and had more pronounced C-type inactivation. These altered gating properties will reduce the delayed-rectifier potassium current which may underlie the symptoms of EA.

Heteromeric channel formation and Ca(2+)-free media reduce the toxic effect of the weaver Kir 3.2 allele.

Weaver mice have a severe hypoplasia of the cerebellum with an almost complete loss of the midline granule cells. Recent genetic studies of weaver mice have identified a mutation resulting in an amino acid substitution (G156S) in the pore of the inwardly rectifying potassium channel subunit Kir 3.2. When expressed in Xenopus oocytes the weaver mutation alters channel selectivity from a potassium-selective to a nonspecific cation-selective pore. In this study we confirm by cell-attached patch-clamp recording that the mutation produces a non-selective cation channel. We also demonstrate that the cell death induced by weaver expression may be prevented by elimination of calcium from the extracellular solution as well as by coexpression with the wild-type Kir 3.2 allele, or other members of the Kir 3.0 subfamily. These results suggest that the weaver defect in Kir 3.2 may cause cerebellar cell death by cell swelling and calcium overload. Cells which express the weaver subunit, but which normally survive, may do so because of heteromeric subunit assembly with wild-type subunits of the Kir 3.0 subfamily.

Calcium and cardiac electrophysiology. Some experimental considerations.

Electrophysiologic experiments in cardiac tissue suggest that Ca2+ is involved in generation of the action potential, the pacemaker potential, and conduction of the slow wave of depolarization. For instance, removal of Ca2+ inhibits the slow inward current and prolongs the action potential and suppresses the slow diastolic depolarization. Divalant cations Mn2+, Co2+, Cd2+, Mg2+, block the slow inward current and suppress pacemaker activity, but shorten the action potential. Ni2+ specifically blocks the slow inward current and prolongs the action potential. Ca2+ also plays a central role in generation of diastolic depolarizaittn. Cd2+ inhibits the diastolic depolarizaton and the upstoke of the action potential in SA nodal cells, while blocking the time-dependent inward current in the pacemaker potential range and the time-dependent outward current. A variety of molecular transport systems ranging from the Ca-channel to a Ca2+-Na+ or Ca2+-K+ exchanges to Ca2+-induced activation of the K+ current have been postulated to explain the effects of Ca2+ on cardiac electrophysiologic processes.

Small-conductance calcium-activated potassium channels.

SK channels play a fundamental role in all excitable cells. SK channels are potassium selective and are activated by an increase in the level of intracellular calcium, such as occurs during an action potential. Their activation causes membrane hyperpolarization, which inhibits cell firing and limits the firing frequency of repetitive action potentials. The intracellular calcium increase evoked by action potential firing decays slowly, allowing SK channel activation to generate a long-lasting hyperpolarization termed the slow afterhyperpolarization (sAHP). This spike-frequency adaptation protects the cell from the deleterious effects of continuous tetanic activity and is essential for normal neurotransmission. Slow AHPs can be classified into two groups, based on sensitivity to the bee venom toxin apamin. In general, apamin-sensitive sAHPs activate rapidly following a single action potential and decay with a time constant of approximately 150 ms. In contrast, apamin-insensitive sAHPs rise slowly and decay with a time constant of approximately 1.5 s. The basis for this kinetic difference is not yet understood. Apamin-sensitive and apamin-insensitive SK channels have recently been cloned. This chapter will compare with different classes of sAHPs, discuss the cloned SK channels and how they are gated by calcium ions, describe the molecular basis for their different pharmacologies, and review the possible role of SK channels in several pathological conditions.

Imidazoline compounds inhibit KATP channels in guinea pig ventricular myocytes.

Phentolamine and related imidazolines inhibit KATP channel activity in the pancreatic beta cell. In the present study, the effects of several imidazoline-based compounds were examined upon KATP channel activity in guinea pig ventricular myocytes. Phentolamine produced a potent inhibition of KATP channel activity when examined in either excised inside-out patches or in the whole-cell configuration. This effect was unrelated to phentolamine's ability to antagonise alpha-adrenoceptors since the nonselective alpha-adrenoceptor antagonists, benextramine and phenoxybenzamine, failed to affect channel activity. Furthermore, the alpha-adrenoceptor agonist clonidine together with several related imidazolines inhibited channel activity. This suggests that imidazoline compounds modulate KATP channel activity in guinea pig ventricular myocytes and this may have clinical implications for the use of such agents as hypoglycemic drugs.