Differential expression of voltage-gated calcium channels in identified visual cortical neurons.

Using the whole-cell patch-clamp technique, Ca2+ channel currents were examined in three distinct types of neurons derived from rat primary visual cortex. Callosal-projecting and superior colliculus-projecting neurons were identified following in vivo retrograde labeling with fluorescent "beads." A subset of intrinsic GABAergic visual cortical neurons was identified with the monoclonal antibody VC1.1. Although high voltage-activated Ca2+ channel currents were measured in all three cell types, clear differences in the densities of these channels were observed. There were also marked variations in the relative amplitudes of the inactivating and noninactivating components of the high voltage-activated currents, suggesting that N- and L-type Ca2+ channels are differentially distributed. Although low voltage-activated or T-type currents were measured in subsets of both types of projection neurons, they were not observed in VC1.1-positive cells. These results provide a direct demonstration that voltage-gated Ca2+ channels are expressed in neurons of the mammalian visual cortex and reveal that the distribution and densities of different Ca2+ channel types in diverse classes of visual cortical neurons are distinct.

Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.

We generated transgenic mice in which red, green, yellow, or cyan fluorescent proteins (together termed XFPs) were selectively expressed in neurons. All four XFPs labeled neurons in their entirety, including axons, nerve terminals, dendrites, and dendritic spines. Remarkably, each of 25 independently generated transgenic lines expressed XFP in a unique pattern, even though all incorporated identical regulatory elements (from the thyl gene). For example, all retinal ganglion cells or many cortical neurons were XFP positive in some lines, whereas only a few ganglion cells or only layer 5 cortical pyramids were labeled in others. In some lines, intense labeling of small neuronal subsets provided a Golgi-like vital stain. In double transgenic mice expressing two different XFPs, it was possible to differentially label 3 neuronal subsets in a single animal.

Caged compounds: tools for illuminating neuronal responses and connections.

A number of new 'caged' intracellular second messengers and neurotransmitters have been developed using the photolabile o-nitrobenzyl group. This chemistry has also recently been exploited in novel ways, including the development of caged enzyme substrates and caged proteins. Although caged compounds continue to be used primarily for mechanistic (kinetic) studies of processes mediated by transmitters or second messengers, the spatial resolution afforded by the use of light to effect changes in transmitter concentrations has now been clearly demonstrated. The increased availability of caged compounds and of the technologies required to exploit them provides neurobiologists with powerful tools for probing neuronal response properties and connectivity patterns.

Genetic manipulation of cardiac K(+) channel function in mice: what have we learned, and where do we go from here?

In the mammalian myocardium, potassium (K(+)) channels control resting potentials, action potential waveforms, automaticity, and refractory periods and, in most cardiac cells, multiple types of K(+) channels that subserve these functions are expressed. Molecular cloning has revealed the presence of a large number of K(+) channel pore forming (alpha) and accessory (beta) subunits in the heart, and considerable progress has been made recently in defining the relationships between expressed K(+) channel subunits and functional cardiac K(+) channels. To date, more than 20 mouse models with altered K(+) channel expression/functioning have been generated using dominant-negative transgenic and targeted gene deletion approaches. In several instances, the genetic manipulation of K(+) channel subunit expression has revealed the role of specific K(+) channel subunit subfamilies or individual K(+) channel subunit genes in the generation of myocardial K(+) channels. In other cases, however, the phenotypic consequences have been unexpected. This review summarizes what has been learned from the in situ genetic manipulation of cardiac K(+) channel functioning in the mouse, discusses the limitations of the models developed to date, and explores the likely directions of future research.

Targeted replacement of KV1.5 in the mouse leads to loss of the 4-aminopyridine-sensitive component of I(K,slow) and resistance to drug-induced qt prolongation.

The K(+) channel mKv1.5 is thought to encode a 4-aminopyridine (4-AP)-sensitive component of the current I(K,slow) in the mouse heart. We used gene targeting to replace mKv1.5 with the 4-AP-insensitive channel rKv1.1 (SWAP mice) and directly test the role of Kv1.5 in the mouse ventricle. Kv1.5 RNA and protein were undetectable, rKv1.1 was expressed, and Kv2.1 protein was upregulated in homozygous SWAP hearts. The density of the K(+) current I(K,slow) (depolarizations to +40 mV, pA/pF) was similar in left ventricular myocytes isolated from SWAP homozygotes (17+/-1, n=27) and littermate controls (16+/-2, n=19). The densities and properties of I(peak), I(to,f), I(to,s), and I(ss) were also unchanged. In homozygous SWAP myocytes, the 50-micromol/L 4-AP-sensitive component of IK,slowwas absent (n=6), the density of the 20-mmol/L tetraethylammonium-sensitive component of I(K,slow) was increased (9+/-1 versus 5+/-1, P<0.05), and no 100- to 200-nmol/L alpha-dendrotoxin-sensitive current was found (n=8). APD(90) in SWAP myocytes was similar to controls at baseline but did not prolong in response to 30 micromol/L 4-AP. Similarly, QTc (ms) was not prolonged in anesthetized SWAP mice (64+/-2, homozygotes, n=9; 62+/-2, controls, n=9), and injection with 4-AP prolonged QTc only in controls (63+/-1, homozygotes; 72+/-2, controls; P<0.05). SWAP mice had no increase in arrhythmias during ambulatory telemetry monitoring. Thus, Kv1.5 encodes the 4-AP-sensitive component of I(K,slow) in the mouse ventricle and confers sensitivity to 4-AP-induced prolongation of APD and QTC: Compensatory upregulation of Kv2.1 may explain the phenotypic differences between SWAP mice and the previously described transgenic mice expressing a truncated dominant-negative Kv1.1 construct.

Molecular heterogeneity of the voltage-gated fast transient outward K+ current, I(Af), in mammalian neurons.

Recently, we identified four kinetically distinct voltage-gated K(+) currents, I(Af), I(As), I(K), and I(SS), in rat superior cervical ganglion (SCG) neurons and demonstrated that I(Af) and I(As) are differentially expressed in type I (I(Af), I(K), I(SS)), type II (I(Af), I(As), I(K), I(SS)), and type III (I(K), I(SS)) SCG cells. In addition, we reported that I(Af) is eliminated in most ( approximately 70%) SCG cells expressing Kv4.2W362F, a Kv4 subfamily-specific dominant negative. The molecular correlate(s) of the residual I(Af), as well as that of I(As), I(K), and I(SS), however, are unknown. The experiments here were undertaken to explore the role of Kv1 alpha-subunits in the generation of voltage-gated K(+) currents in SCG neurons. Using the Biolistics Gene Gun, cDNA constructs encoding a Kv1 subfamily-specific dominant negative, Kv1.5W461F, and enhanced green fluorescent protein (EGFP) were introduced into SCG neurons. Whole-cell recordings from EGFP-positive Kv1.5W461F-expressing cells revealed a selective decrease in the percentage of type I cells and an increase in type III cells, indicating that I(Af) is gated by Kv1 alpha-subunits in a subset of type I SCG neurons. I(Af) is eliminated in all SCG cells expressing both Kv1.5W461F and Kv4.2W362F. I(Af) tau(decay) values in Kv1.5W461F-expressing and Kv4.2W362F-expressing type I cells are significantly different, revealing that Kv1 and Kv4 alpha-subunits encode kinetically distinct I(Af) channels. Expression of Kv1.5W461F increases excitability by decreasing action potential current thresholds and converts phasic cells to adapting or tonic firing. Interestingly, the molecular heterogeneity of I(Af) channels has functional significance because Kv1- and Kv4-encoded I(Af) play distinct roles in the regulation of neuronal excitability.

Two functionally distinct 4-aminopyridine-sensitive outward K+ currents in rat atrial myocytes.

In the experiments here, the detailed kinetic properties of the Ca(2+)-independent, depolarization-activated outward currents (Iout) in enzymatically dispersed adult rat atrial myocytes were studied. Although there is only slight attenuation of peak Iout during brief (100 ms) voltage steps, substantial decay is evident during long (10 s) depolarizations. The analyses here reveal that current inactivation is best described by the sum of two exponential components, which we have termed IKf and IKs to denote the fast and slow components, respectively, of Iout decay. At all test potentials, IKf inactivates approximately 20-fold more rapidly than IKs. Neither the decay time constants nor the fraction of Iout remaining at the end of 10-s depolarizations varies over the potential range of 0 to +50 mV, indicating that the rates of inactivation and recovery from inactivation are voltage independent. IKf recovers from inactivation completely, independent of the recovery of IKs, and IKf recovers approximately 20 times faster than IKs. The pharmacological properties of IKf and IKs are similar: both components are sensitive to 4-aminopyridine (1-5 mM) and both are relatively resistant to externally applied tetraethylammonium (50 mM). Taken together, these findings suggest that IKf and IKs correspond to two functionally distinct K+ currents with similar voltage-dependent properties and pharmacologic sensitivities, but with markedly different rates of inactivation and recovery from inactivation. From the experimental data, several gating models were developed in which voltage-independent inactivation is coupled either to channel opening or to the activation of the individual channel subunits. Experimental testing of predictions of these models suggests that voltage-independent inactivation is coupled to activation, and that inactivation of only a single subunit is required to result in functional inactivation of the channels. This model closely approximates the properties of IKf and IKs, as well as the composite outward currents, measured in adult rat atrial myocytes.

Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes.

Depolarization-activated outward K+ currents in isolated adult rat ventricular myocytes were characterized using the whole-cell variation of the patch-clamp recording technique. During brief depolarizations to potentials positive to -40 mV, Ca(2+)-independent outward K+ currents in these cells rise to a transient peak, followed by a slower decay to an apparent plateau. The analyses completed here reveal that the observed outward current waveforms result from the activation of two kinetically distinct voltage-dependent K+ currents: one that activates and inactivates rapidly, and one that activates and inactivates slowly, on membrane depolarization. These currents are referred to here as Ito (transient outward) and IK (delayed rectifier), respectively, because their properties are similar (although not identical) to these K+ current types in other cells. Although the voltage dependences of Ito and IK activation are similar, Ito activates approximately 10-fold and inactivates approximately 30-fold more rapidly than IK at all test potentials. In the composite current waveforms measured during brief depolarizations, therefore, the peak current predominantly reflects Ito, whereas IK is the primary determinant of the plateau. There are also marked differences in the voltage dependences of steady-state inactivation of these two K+ currents: IK undergoes steady-state inactivation at all potentials positive to -120 mV, and is 50% inactivated at -69 mV; Ito, in contrast, is insensitive to steady-state inactivation at membrane potentials negative to -50 mV. In addition, Ito recovers from steady-state inactivation faster than IK: at -90 mV, for example, approximately 70% recovery from the inactivation produced at -20 mV is observed within 20 ms for Ito; IK recovers approximately 25-fold more slowly. The pharmacological properties of Ito and IK are also distinct: 4-aminopyridine preferentially attenuates Ito, and tetraethylammonium suppresses predominantly IK. The voltage- and time-dependent properties of these currents are interpreted here in terms of a model in which Ito underlies the initial, rapid repolarization phase of the action potential (AP), and IK is responsible for the slower phase of AP repolarization back to the resting membrane potential, in adult rat ventricular myocytes.

Four kinetically distinct depolarization-activated K+ currents in adult mouse ventricular myocytes.

In the experiments here, the time- and voltage-dependent properties of the Ca2+-independent, depolarization-activated K+ currents in adult mouse ventricular myocytes were characterized in detail. In the majority (65 of 72, approximately 90%) of cells dispersed from the ventricles, analysis of the decay phases of the outward currents revealed three distinct K+ current components: a rapidly inactivating, transient outward K+ current, Ito,f (mean +/- SEM taudecay = 85 +/- 2 ms); a slowly (mean +/- SEM taudecay = 1,162 +/- 29 ms) inactivating K+ current, IK,slow; and a non inactivating, steady state current, Iss. In a small subset (7 of 72, approximately 10%) of cells, Ito,f was absent and a slowly inactivating (mean +/- SEM taudecay = 196 +/- 7 ms) transient outward current, referred to as Ito,s, was identified; the densities and properties of IK,slow and Iss in Ito,s-expressing cells are indistinguishable from the corresponding currents in cells with Ito,f. Microdissection techniques were used to remove tissue pieces from the left ventricular apex and from the ventricular septum to allow the hypothesis that there are regional differences in Ito,f and Ito,s expression to be tested directly. Electrophysiological recordings revealed that all cells isolated from the apex express Ito,f (n = 35); Ito,s is not detected in these cells (n = 35). In the septum, by contrast, all of the cells express Ito,s (n = 28) and in the majority (22 of 28, 80%) of cells, Ito,f is also present. The density of Ito,f (mean +/- SEM at +40 mV = 6.8 +/- 0.5 pA/pF, n = 22) in septum cells, however, is significantly (P < 0.001) lower than Ito,f density in cells from the apex (mean +/- SEM at +40 mV = 34.6 +/- 2.6 pA/pF, n = 35). In addition to differences in inactivation kinetics, Ito,f, Ito,s, and IK,slow display distinct rates of recovery (from inactivation), as well as differential sensitivities to 4-aminopyridine (4-AP), tetraethylammonium (TEA), and Heteropoda toxin-3. IK,slow, for example, is blocked selectively by low (10-50 microM) concentrations of 4-AP and by (>/=25 mM) TEA. Although both Ito,f and Ito,s are blocked by high (>100 microM) 4-AP concentrations and are relatively insensitive to TEA, Ito,f is selectively blocked by nanomolar concentrations of Heteropoda toxin-3, and Ito,s (as well as IK,slow and Iss) is unaffected. Iss is partially blocked by high concentrations of 4-AP or TEA. The functional implications of the distinct properties and expression patterns of Ito,f and Ito,s, as well as the likely molecular correlates of these (and the IK,slow and Iss) currents, are discussed.

Photoinduced removal of nifedipine reveals mechanisms of calcium antagonist action on single heart cells.

The currents through voltage-activated calcium channels in heart cell membranes are suppressed by dihydropyridine calcium antagonists such as nifedipine. Nifedipine is photolabile, and the reduction of current amplitude by this drug can be reversed within a few milliseconds after a 1-ms light flash. The blockade by nifedipine and its removal by flashes were studied in isolated myocytes from neonatal rat heart using the whole-cell clamp method. The results suggest that nifedipine interacts with closed, open, and inactivated calcium channels. It is likely that at the normal resting potential of cardiac cells, the suppression of current amplitude arises because nifedipine binds to and stabilizes channels in the resting, closed state. Inhibition is enhanced at depolarized membrane potentials, where interaction with inactivated channels may also become important. Additional block of open channels is suggested when currents are carried by Ba2+ but is not indicated with Ca2+ currents. Numerical simulations reproduce the experimental observations with molecular dissociation constants on the order of 10(-7) M for closed and open channels and 10(-8) M for inactivated channels.

Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes.

In the experiments here, the developmental expression of the functional Ca(2+)-independent, depolarization-activated K+ channel currents, Ito and IK, and of the voltage-gated K+ channel (Kv) alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2 in rat ventricular myocytes were examined quantitatively. Using the whole-cell patch clamp recording method, the properties and the densities of Ito and IK in ventricular myocytes isolated from postnatal day 5 (P5), 10 (P10), 15 (P15), 20 (P20), 25 (P25), 30 (P30), and adult (8-12 wk) rats were characterized and compared. These experiments revealed that mean Ito densities increase fourfold between birth and P30, whereas IK densities vary only slightly. Neither the time- nor the voltage-dependent properties of the currents vary measurably, suggesting that the subunits underlying functional Ito and IK channels are the same throughout postnatal development. In parallel experiments, the developmental expression of each of the voltage-gated K+ channel alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, was examined quantitatively at the mRNA and protein levels using subunit-specific probes. RNase protection assays revealed that Kv1.4 message levels are high at birth, increase between P0 and P10, and subsequently decrease to very low levels in adult rat ventricles. The decrease in message is accompanied by a marked reduction in Kv1.4 protein, consistent with our previous suggestion that Kv1.4 does not contribute to the formation of functional K+ channels in adult rat ventricular myocytes. In contrast to Kv1.4, the mRNA levels of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 increase (three- to five-fold) between birth and adult. Western analyses, however, revealed that the expression patterns of these subunits proteins vary in distinct ways: Kv1.2 and Kv4.2, for example, increase between P5 and adult, whereas Kv1.5 remains constant and Kv2.1 decreases. Throughout development, therefore, there is a mismatch between the numbers of Kv alpha subunits expressed and the functional voltage-gated K+ channel currents distinguished electrophysiologically in rat ventricular myocytes. Alternative experimental approaches will be required to define directly the Kv alpha subunits that underlie functional voltage-gated K+ channels in these (and other) cells. In addition, the finding that Kv alpha subunit protein expression levels do not necessarily mirror mRNA levels suggests that caution should be exercised in attempting functional interpretations of observed changes in mRNA levels alone.

Distinct transient outward potassium current (Ito) phenotypes and distribution of fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes.

The biophysical characteristics and alpha subunits underlying calcium-independent transient outward potassium current (Ito) phenotypes expressed in ferret left ventricular epicardial (LV epi) and endocardial (LV endo) myocytes were analyzed using patch clamp, fluorescent in situ hybridization (FISH), and immunofluorescent (IF) techniques. Two distinct Ito phenotypes were measured (21-22 degrees C) in the majority of LV epi and LV endo myocytes studied. The two Ito phenotypes displayed marked differences in peak current densities, activation thresholds, inactivation characteristics, and recovery kinetics. Ito,epi recovered rapidly [taurec, -70 mV = 51 +/- 3 ms] with minimal cumulative inactivation, while Ito,endo recovered slowly [taurec, -70 mV = 3,002 +/- 447 ms] with marked cumulative inactivation. Heteropoda toxin 2 (150 nM) blocked Ito,epi in a voltage-dependent manner, but had no effect on Ito,endo. Parallel FISH and IF measurements conducted on isolated LV epi and LV endo myocytes demonstrated that Kv1.4, Kv4.2, and Kv4.3 alpha subunit expression in LV myocyte types was quite heterogenous: (a) Kv4.2 and Kv4.3 were more predominantly expressed in LV epi than LV endo myocytes, and (b) Kv1.4 was expressed in the majority of LV endo myocytes but was essentially absent in LV epi myocytes. In combination with previous measurements on recovery kinetics (Kv1.4, slow; Kv4.2/4.3, relatively rapid) and Heteropoda toxin block (Kv1.4, insensitive; Kv4.2, sensitive), our results strongly support the hypothesis that, in ferret heart, Kv4.2/Kv4.3 and Kv1.4 alpha subunits, respectively, are the molecular substrates underlying the Ito,epi and Ito,endo phenotypes. FISH and IF measurements were also conducted on ferret ventricular tissue sections. The three Ito alpha subunits again showed distinct patterns of distribution: (a) Kv1.4 was localized primarily to the apical portion of the LV septum, LV endocardium, and approximate inner 75% of the LV free wall; (b) Kv4. 2 was localized primarily to the right ventricular free wall, epicardial layers of the LV, and base of the heart; and (c) Kv4.3 was localized primarily to epicardial layers of the LV apex and diffusely distributed in the LV free wall and septum. Therefore, in intact ventricular tissue, a heterogeneous distribution of candidate Ito alpha subunits not only exists from LV epicardium to endocardium but also from apex to base.

ERG proteins and functional cardiac I(Kr) channels in rat, mouse, and human heart.

The voltage-gated K(+) channel (Kv) pore forming alpha subunit, ERG1 (KCNH2), has been identified as the locus of mutations in one type of inherited long QT syndrome, LQT2. Heterologous expression of ERG1 reveals rapidly activating and inactivating K(+) currents, characterized by marked inward rectification at potentials positive to 0 mV, which are similar to the rapid component of cardiac delayed rectification I(Kr). There are, however, marked differences in the properties of expressed ERG1 and endogenous cardiac I(Kr), suggesting that functional I(Kr) channels reflect the coassembly of full-length ERG1 with splice variants and /or accessory subunits. Consistent with these hypotheses, N- and C-terminal variants of ERG1 have been identified, and it has been demonstrated that heterologously expressed ERG1 and minK (or MiRP1) coimmunoprecipitate. Recent biochemical studies, however, suggest that only full-length ERG1 is expressed in adult mouse, rat, or human heart. Clearly, further studies, focused on identifying the subunits that coassemble with ERG1 in vivo, as well as on post-translational processing of the full-length ERG1 protein will be necessary to define the molecular composition of functional cardiac I(Kr) channels.

Direct evidence for the inhibition of platelet aggregation and release by intracellular cyclic AMP produced with a new photoactivatable derivative.

An increase in platelet cyclic AMP (cAMP) via stimulation of adenylate cyclase is thought to be the underlying mechanism by which potent prostaglandins i.e. PGD2, PGI2, inhibit platelet functions. We report here new and direct evidence for the inhibitory effects of cAMP on platelet aggregation and serotonin release. Washed platelets from rat were incubated with a new photoactivatable cAMP analogue (4,5-dimethoxy-2-nitrobenzyl ester); this compound is almost physiologically inert before irradiation and liberates free cAMP ("cAMP jumps") following light flashes. A single flash, delivered after 2 min incubation in 100-200 microM of the analogue, dramatically inhibited thrombin-induced aggregation, as compared with controls. Endogenous serotonin release, measured in the same samples by means of an electrochemically treated carbon electrode was undetectable after the cAMP jump. Pre-irradiated solutions added to platelets had no effect. The kinetics of the flash-induced effects were also studied. From these results we can conclude that: i) the photoactivatable cAMP derivative has to permeate through the platelet membrane; ii) the analogue remains photolabile; and, iii) intracellular cAMP, resulting from photolysis dramatically inhibits platelet aggregation and serotonin release. It is possible that cAMP exerts its effects by regulating cytoplasmic free calcium concentration and/or other actions affecting platelet activation.

Development of the fast, transient outward K+ current in embryonic sympathetic neurones.

Voltage-activated outward potassium (K+) currents in developing sympathetic neurones, dissociated from the rat superior cervical ganglion (SCG), were studied using the whole-cell patch clamp recording technique. In voltage-clamped neonatal SCG cells, two voltage-dependent K+ currents were measured: the fast, transient K+ current, IA; and, the slower activating, non-inactivating delayed rectifier, IK. Only IK, however, appeared to be present in SCG neurones isolated from early embryonic (E14.5-16.5) rat pups; IA was not observed in these cells. When these embryonic neurones were maintained in cell culture, IA developed over a time course (approximately 4-6 days) similar to that seen in vivo. IA, therefore, which appears to facilitate the fast repolarization phase of the action potential in rat SCG neurones, is the last voltage-activated current to develop in these cells.

Photolabile "caged" adrenergic receptor agonists and related model compounds.

The synthesis and photochemical characterization of caged derivatives of the adrenergic receptor agonists phenylephrine, epinephrine and isoproterenol are described. These compounds were prepared using 2-nitrobenzyl or substituted 2-nitrobenzyl photolabile protecting groups, and were designed to allow agonist concentration jumps to be made during pharmacological/physiological experiments. The advantage of this approach over conventional methods for changing the concentrations of agonists near receptors in mechanistic studies is the exquisite spatial and temporal resolution afforded by the use of light. Flash photolysis experiments revealed that photorelease is more than two orders of magnitude faster when the 2-nitrobenzyl group is attached to the beta-amino group rather than one of the phenolic oxygens of the catecholamine. For the caged phenylephrine derivatives, for example, the rate constants of release from the N-linked and O-linked derivatives are 1.8 x 10(4) s-1 and 1.1 x 10(2) s-1 respectively. However, the quantum yields of photorelease from the N-linked and O-linked derivatives are similar. In addition, several model compounds were prepared to allow examination of the effects of substituents on the aromatic ring and benzylic carbon (of the 2-nitrobenzyl moiety) on the rates and efficiencies of photorelease. These studies revealed that, although substituents had little effect on the rates of photorelease from the N-linked caged derivatives, electron-donating groups on the 2-nitrobenzyl ring increased the quantum yield of release by approximately fourfold, from 0.10 to 0.40. A summary of the studies completed to evaluate the biological properties of the caged adrenergic receptor agonists is also presented.

Vascular actions of 'caged' phenylephrine analogs depend on the structure and site of attachment of the 2-nitrobenzyl group.

In the experiments presented in this article, the effects of four caged analogs of the alpha 1-adrenergic agonist phenylephrine (PE) on the properties of small (100-200 microns outer diameter), isolated rat mesenteric arteries were compared. The four caged PE analogs contained either an unsubstituted (analogs I and II) or an alpha-carboxy substituted (analogs III and IV) 2-nitrobenzyl group attached to the phenolic oxygen atom (O-linked; analogs II and IV) or to the amino group (N-linked; analogs I and III) of PE. The structure of each caged PE analog was confirmed by UV, IR and 1H NMR spectral analysis. For physiological experiments, photolysis of the caged PE analogs was accomplished with a Hi-Tech Scientific flashlamp, and vascular smooth muscle contraction was measured with a computer-based image analysis system. In some experiments, the fura-2 ratiometric technique was used to examine the effects of the caged PE analogs on intracellular Ca2+ levels. At concentration < or = 10(-6) M, none of the four analogs displayed measurable intrinsic vasoconstricting activity, that is, vasoconstrictions were only observed following light flashes, consistent with the release of free PE. At concentrations > or = 10(-5) M, however, both O-linked compounds (analogs II and IV) and the alpha-carboxy substituted N-linked caged PE (analog III) produced vasoconstriction prior to photolysis. In contrast, no intrinsic vasoconstricting activity was evident with the unsubstituted N-linked caged PE (analog I) at concentrations up to 300 microM (the highest concentration tested). At concentrations > or = 10 microM, the O-linked unsubstituted caged PE (analog II) also had intrinsic vasodilating activity and markedly attenuated vasoconstrictions and increases in intracellular Ca2+ produced by high KCl. Similar effects were observed with the N-linked caged PE analogs (I and III) at > or = 100 microM, whereas no measurable relaxations were seen with the alpha-carboxy O-linked caged PE analog (i.v.) at concentrations up to 300 microM (the highest concentration tested). Taken together, the results presented here demonstrate that the N-linked unsubstituted caged PE analog (I) can be used reliably at concentrations up to 100 microM and is, therefore, the analog of choice for physiological studies of alpha 1-receptor-mediated events.

Deletion of the Kv2.1 delayed rectifier potassium channel leads to neuronal and behavioral hyperexcitability.

The Kv2.1 delayed rectifier potassium channel exhibits high-level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity-dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1(-/-) ) mice lacking this channel. Kv2.1(-/-) mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1(-/-) mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1(-/-) mice appear unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1(-/-) animals. Field recordings from hippocampal slices of Kv2.1(-/-) mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1(-/-) mice, long-term potentiation at the Schaffer collateral - CA1 synapse is decreased. Kv2.1(-/-) mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1(-/-) mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1(-/-) mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function.