A progressive assessment strategy improves student learning and perceived course quality in undergraduate physiology.

In 2010, second-year physiology (n = 165) had a traditional single 3-h end-of-semester exam. To provide diagnostic feedback earlier, for students enrolled in 2011 (n = 128), we incorporated an in-class exam at 3 wk in addition to the final exam. Based on initial analysis and positive student comments, for the 2012 cohort (n = 148), we expanded this to incorporate four 1-h in-class exams every 3 wk plus a short final integrative exam. Average scores from exams and questionnaires (student evaluations of learning and teaching, 10 questions) were compared among 2010, 2011, and 2012 cohorts. We also compared scores in the practical component of the course, which had a constant format for all cohorts. Data are given as means ± SD; statistical analyses were done with unpaired two-way Students t-tests. From 2010 to 2012, there was a significant improvement in total exam scores (59.7 ± 15.8 vs. 68.6 ± 14.2, P < 0.001) but no significant change in total practical scores (72.3 ± 9.0 vs. 74.4 ± 10.2, P = 0.05), indicating that the rise in exam score was not due to higher academic abilities of the 2012 cohort. Overall mean student evaluation of learning and teaching responses (4.9 ± 0.4 vs. 5.3 ± 0.3, P = 0.015) and overall percent broad agreement (66.0 ± 8.0 vs. 79.2 ± 7.5, P = 0.003) indicated a significant improvement in student satisfaction. In conclusion, both learning outcome and perceived course quality were enhanced by the increased frequency of examinations, possibly by promoting consistent student study habits.

Role of aquaporin-1 in trabecular meshwork cell homeostasis during mechanical strain.

Aquaporin-1 (AQP1) channels are expressed by trabecular meshwork (TM) and Schlemm's canal cells of the conventional outflow pathway where fluid movement is predominantly paracellular, suggesting a non-canonical role for AQP1. We hypothesized that AQP1 functions to protect TM cells during periods of mechanical strain. To test this idea, primary cultures of confluent human TM cells on Bioflex membranes were exposed to static and cyclic stretch for 8 and 24h using the Flexcell system. AQP1 expression in TM cells was assessed by SDS-PAGE and Western blot using anti-AQP1 IgGs. AQP1 protein bands were analyzed using densitometry and normalized to beta-actin expression. Cell damage was monitored by measuring lactate dehydrogenase (LDH) and histone deacetylase appearance in conditioned media. Recombinant expression of AQP1 in TM cell cultures was facilitated by transduction with adenovirus. Results show that AQP1 expression significantly increased 2-fold with 10% static stretch and 3.5-fold with 20% static stretch at 8h (n=4, p<0.05) and 24h (n=6, p<0.05). While histone deacetylase levels were unaffected by treatments, release of LDH from TM cells was the most profound at the 20% static stretch level (n=4, p<0.05). Significantly, cells were refractory to the 20% static stretch level when AQP1 expression was increased to near tissue levels. Analysis of LDH release with respect to AQP1 expression revealed an inverse linear relationship (r(2)=0.7780). Taken together, AQP1 in human TM appears to serve a protective role by facilitating improved cell viability during conditions of mechanical strain.

Forskolin stimulation of water and cation permeability in aquaporin 1 water channels.

Aquaporin 1, a six-transmembrane domain protein, is a water channel present in many fluid-secreting and -absorbing cells. In Xenopus oocytes injected with aquaporin 1 complementary RNA, the application of forskolin or cyclic 8-bromo- adenosine 3',5'-monophosphate increased membrane permeability to water and triggered a cationic conductance. The cationic conductance was also induced by direct injection of protein kinase A (PKA) catalytic subunit, reduced by the kinase inhibitor H7, and blocked by HgCl2, an inhibitor of aquaporin 1. The cationic permeability of the aquaporin 1 channel is activated by a cyclic adenosine monophosphate-dependent mechanism that may involve direct or indirect phosphorylation by PKA.

Fractional Deletion of Compound Kushen Injection Indicates Cytokine Signaling Pathways are Critical for its Perturbation of the Cell Cycle.

We used computational and experimental biology approaches to identify candidate mechanisms of action of aTraditional Chinese Medicine, Compound Kushen Injection (CKI), in a breast cancer cell line (MDA-MB-231). Because CKI is a complex mixture of plant secondary metabolites, we used a high-performance liquid chromatography (HPLC) fractionation and reconstitution approach to define chemical fractions required for CKI to induce apoptosis. The initial fractionation separated major from minor compounds, and it showed that major compounds accounted for little of the activity of CKI. Furthermore, removal of no single major compound altered the effect of CKI on cell viability and apoptosis. However, simultaneous removal of two major compounds identified oxymatrine and oxysophocarpine as critical with respect to CKI activity. Transcriptome analysis was used to correlate compound removal with gene expression and phenotype data. Many compounds in CKI are required to trigger apoptosis but significant modulation of its activity is conferred by a small number of compounds. In conclusion, CKI may be typical of many plant based extracts that contain many compounds in that no single compound is responsible for all of the bioactivity of the mixture and that many compounds interact in a complex fashion to influence a network containing many targets.

Over-expression of the potassium channel Kir2.3 using the dopamine-1 receptor promoter selectively inhibits striatal neurons.

Dysfunction of basal ganglia circuits underlies a variety of movement disorders and neuropsychiatric conditions. Selective control of the electrical activity of striatal outflow pathways by manipulation of ion channel function presents a novel therapeutic approach. Toward this end, we have constructed and studied in vitro an adenoviral gene transfer vector that employs the promoter region of the dopamine-1 receptor to drive expression of the inward rectifier K(+) channel Kir2.3. The use of this neuronal promoter confers cell-type specificity and a physiological level of trans-gene expression in rat primary striatal cultures. The electrophysiological properties were confirmed in transfected human embryonic kidney cells, in which an inwardly-rectifying, Cs(+)-sensitive current was measured by voltage clamp. Current clamp studies of transduced striatal neurons demonstrated an increase in the firing threshold, latency to first action potential and decrease in neuronal excitability. Neurotoxin-induced activation of c-Fos, a marker of neuronal activity, was blocked in transduced neurons indicating that the decrease in electrical excitability was physiologically significant. When used in vivo, this strategy may have the potential to positively impact movement disorders by selectively changing activity of neurons belonging to the direct striatal pathway, characterized by the expression of dopamine-1 receptors.

Simulating pathways of subsurface oil in the Faroe-Shetland Channel using an ocean general circulation model.

Little is known about the fate of subsurface hydrocarbon plumes from deep-sea oil well blowouts and their effects on processes and communities. As deepwater drilling expands in the Faroe-Shetland Channel (FSC), oil well blowouts are a possibility, and the unusual ocean circulation of this region presents challenges to understanding possible subsurface oil pathways in the event of a spill. Here, an ocean general circulation model was used with a particle tracking algorithm to assess temporal variability of the oil-plume distribution from a deep-sea oil well blowout in the FSC. The drift of particles was first tracked for one year following release. Then, ambient model temperatures were used to simulate temperature-mediated biodegradation, truncating the trajectories of particles accordingly. Release depth of the modeled subsurface plumes affected both their direction of transport and distance travelled from their release location, and there was considerable interannual variability in transport.

Cloning of a receptor for prostaglandin F2 alpha from the ovine corpus luteum.

A complementary DNA clone encoding a functional receptor for prostaglandin F2 alpha (PGF2 alpha) has been isolated from an ovine large luteal cell complementary DNA library (prepared from day 10 mid-luteal phase RNA). This receptor, which has been designated FP, consists of 362 amino acids (M(r) = 40,982) and is a member of the family of G protein-coupled receptors. Radioligand binding studies with membranes prepared from transfected COS cells demonstrated specific 17-[3H]phenyl-trinor-PGF2 alpha binding that was displaced by prostanoids in the order of 17-phenyl-trinor-PGF2 alpha > PGF2 alpha > fluprostenol > PGD2 > PGE2 >> 8-epi PGF2 alpha. Xenopus laevis oocytes injected with RNA encoding the ovine FP receptor responded to 17-phenyl-trinor-PGF2 alpha with increased membrane chloride conductance in calcium-free medium. Northern blot analysis with RNA from day 10 corpus luteum showed a major band of approximately 6.1 kilobases. On day 14, when luteolysis usually starts, the abundance of this 6.1-kilobase band was variable between individual ewes, and on day 16, when luteolysis is underway, the message was uniformly less abundant. This variability appeared to correlate with circulating progesterone. Thus, when the progesterone level was high (days 10 and 14 depending on whether luteolysis had started), the amount of FP receptor message was high, whereas when the progesterone level was low or falling (day 16), the amount of FP receptor message decreased. We have cloned an ovine FP receptor whose expression confers appropriate functional activity in COS cells and Xenopus oocytes. Furthermore, the level of messenger RNA encoding the FP receptor is high in the midluteal phase ovine corpus luteum and decreases during luteolysis.

Developmental regulation of the A-type potassium-channel current in hippocampal neurons: role of the Kvbeta 1.1 subunit.

The rapidly inactivating A-type K+ current (IA) is prominent in hippocampal neurons; and the speed of its inactivation may regulate electrical excitability. The auxiliary K+ channel subunit Kvbeta 1.1 confers fast inactivation to Shaker-related channels and is postulated to affect IA. Whole-cell patch clamp recordings of rat hippocampal pyramidal neurons in primary culture showed a developmental decrease in the time constant of inactivation (tau(in)) of voltage-gated K+ currents: 17.9+/-1.5 ms in young neurons (5-7 days in vitro; n=53, mean+/-S.E.M.); 9.9+/-1.0 ms in mature neurons (12-15 days in vitro; n=72, mean+/-S.E.M., P<0.01). During the same developmental time, the level of Kvbeta 1.1 transcript increased more than two-fold in vitro and in vivo, determined by semi-quantitative reverse transcriptase-polymerase chain reaction for hippocampus. The hypothesis that up-regulation of Kvbeta 1.1 led to the changes in tau(in) was tested in vitro, using antisense knockdown. Kvbeta 1.1-specific antisense DNA was introduced with a modified herpes virus co-expressing enhanced green fluorescent protein and knockdown of Kvbeta 1.1 was verified by immunocytochemistry. Following transduction with the antisense virus, mature neurons reverted to tau(in) values characteristic of young neurons: 18.3+/-2.4 ms (n=20). The effect of antisense knockdown on electrical excitability was tested using current-clamp protocols to induce repetitive firing. Treatment with the antisense virus increased the interspike interval over a range of membrane depolarization (baseline membrane potential=-40 to +20 mV). This effect was most pronounced at -40 mV, where the ISI of the first pair of action potentials was nearly doubled. These data indicate that Kvbeta 1.1 contributes to the developmental control of IA in hippocampal neurons and that the magnitude of effect is sufficient to regulate electrical excitability. Viral-mediated antisense knockdown of Kvbeta 1.1 is capable of decreasing the electrical excitability of post-mitotic hippocampal neurons, suggesting this approach has applicability to gene therapy of neurological diseases associated with hyperexcitability.

Development of spontaneous and glutamate-evoked activity is altered by chronic ethanol in cultured cerebellar Purkinje neurons.

The effects of continuous exposure to ethanol on the cytological and physiological development of a central nervous system (CNS) neuron were studied using the cultured Purkinje neuron of the rat cerebellar cortex. Purkinje neurons in fetal rat brain cultures which are established at one day before birth show development comparable to that described in vivo in other studies. In culture, Purkinje neurons progress from immature rounded cells with fine neurites to mature neurons with a branched dendritic structure. These structural changes are accompanied by an increase in the duration and complexity of the excitatory response to glutamate, by transitions in the patterns of spontaneous activity, and by an increase in mean firing rate. Our results demonstrate that chronic exposure to a low concentration of ethanol (90 mg%; 19.5 mM) during development selectively alters the electrophysiological but not the morphological properties of Purkinje neurons. Specifically, ethanol treatment reduces the responsiveness of these neurons to glutamate, delays the expected developmental transitions in patterns of spontaneous activity, and induces increased spontaneous bursting activity, particularly at the stage of dendritic formation. Impairment of responsiveness to glutamate is significant in that it may reflect the compromise by ethanol of a major excitatory pathway in the cerebellar cortex, resulting from the decreased efficacy of glutamatergic input from parallel fibers. In contrast to the results of other studies using adult neurons as a model for the effects of ethanol, our work suggests that the developing CNS neuron does not become tolerant; that is, in the continuing presence of ethanol, it does not express physiological function equivalent to that of the control.

Multiple ionic mechanisms are activated by the potent agonist quisqualate in cultured cerebellar Purkinje neurons.

Current clamp recordings were used to analyze responses of cultured cerebellar Purkinje neurons to quisqualate and several other selective non-N-methyl- D-aspertate (NMDA) agonists. Quisqualate, a potent agonist in the cerebellar Purkinje neuron, evoked both short- and long-term changes in excitability, that activated within seconds and lasted for several minutes. Two components of the response were activated differentially by subtype selective agonists, and differed in their mechanism of expression and time course. The initial component of the response was activated by ionotropic agonists ((RS)-d-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) domoate), and by quisqualate and glutamate which are effective at both the ionotropic and metabotropic quisqualate receptor subtypes, but not by the metabotropic agonist trans (+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD). This component was dependent on extracellular Na+, and characterized by a rapid depolarization with a short latency (less than 1-2 s) and a decrease in membrane resistance as expected for an ionotropic reponse. The rapid depolarization extended into an agonist-dependent plateau phase, which could not be evoked by depolarization alone. The second ('late') phase of the response was a slowly-activating, long-lasting change in membrane excitability, accompanied by little or no change in the membrane potential. The late phase, marked by an increase in voltage-dependent bursting spike activity, was induced by the metabotropic agonist, ACPD, and by quisqualate and glutamate, but not by ionotropic selective agonists such as AMPA. Little or no bursting was evoked by AMPA, domoate, kainate or homocysteate. This late phase was also accompanied by increases in the magnitude and duration of the complex spikes and in the afterhyperpolarization following brief current-driven depolarizations. The slower time course of the late component is consistent with a pathway involving second messenger systems. Our results support the hypothesis that coregulation of both ionotropic and metabotropic mechanisms produces the complex and prolonged excitatory response characteristic of the Purkinje neuron.

Expression of Niemann-Pick type C transcript in rodent cerebellum in vivo and in vitro.

This study assesses the developmental expression of the Niemann-Pick type C mRNA in vivo and in vitro in rat cerebellum. NPC is an autosomal recessive neurovisceral lipid storage disease associated with an alteration in cholesterol trafficking. In the mouse model of NPC and in the early onset form of human NPC, Purkinje neurons are among the first neurological targets, suffering stunted growth during postnatal development and dying, leading to ataxia. Recently, the genes responsible for human (NPC1) and mouse (Npc1) NPC disease have been cloned. Based on a highly homologous domain, we designed primers to look for levels of Npc1 mRNA with a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) approach using cyclophilin as an internal standard. Total RNA was isolated from various postnatal developmental stages of the rat cerebellum as template for the analyses. Npc1 transcripts were observed at postnatal day 0 and at later stages of development, both in vivo and in vitro from primary cerebellar cultures. To identify the location of Npc1 inside the cerebellum, we performed immunostaining with an anti-Npc1 antibody in primary rat cerebellar cultures identifying reactive Purkinje neurons by double-labeling with the Purkinje specific marker calbindin and sub-populations of glial cells. In summary, Npc1 is expressed in rat cerebellum in vivo and in vitro and is expressed during early postnatal development as well as in the adult cerebellum. Since Npc1 is expressed at similar levels throughout development, the vulnerability of Purkinje neurons to this disease is likely to involve disruption of an interaction with other developmentally-regulated proteins.

Morphological consequences of altered calcium-dependent transmembrane signaling on the development of cultured cerebellar Purkinje neurons.

Morphometric analyses of cultured rat Purkinje neurons, visualized with anti-calbindin, demonstrated that elevated KCl (10 mM) significantly increased dendritic outgrowth and branching. The response was blocked by NiCl2 (50 microM; R-type Ca2+ channel antagonist). Cells grown in low external Ca2+ (100 nM) showed no loss of responsiveness to elevated potassium. However, thapsigargin (1 microM; Ca(2+)-ATPase blocker) inhibited dendrite outgrowth, suggesting that intracellular calcium stores may be important in governing development.

Increased calcium-dependent K+ channel activity contributes to the maturation of cellular firing patterns in developing cerebellar Purkinje neurons.

Developmental changes in neuronal excitability reflect the regulated expression of ion channels and receptors. Purkinje neurons of the rat cerebellum progress from slow irregular firing to a fast pacemaker-like pattern during postnatal development in vivo. In this study, a comparable period of development in culture was investigated at the protein level using cell-attached single channel recordings to quantify the abundance of active calcium-dependent (KCa) and delayed rectifier (KD) potassium channels. In control cultures, KCa channel activity increased whereas KD channel activity was not significantly different with developmental age. The increase in active KCa channels was antagonized by chronic treatment with the blocker, tetraethylammonium (TEA, 1 mM), which also retarded the normal development of cellular firing patterns. The consequences of chronic TEA treatment were assessed in cultures after thorough washout of the TEA-containing culture medium. Current clamp analyses (nystatin-perforated patches) showed that control Purkinje neurons progressed from a single spike mode to a repetitive firing mode, with a concomitant decrease in action potential duration and an increase in maximal firing rate. Chronic TEA treatment prevented these changes; Purkinje neurons retained the slow firing rate and long duration action potentials that are typical of the immature state. These data suggest that the developmental increase in KCa channel activity may be required for the maturation of cellular firing patterns in cerebellar Purkinje neurons.

Antisense knockdown of calcium-dependent K+ channels in developing cerebellar Purkinje neurons.

Normal developmental upregulation of K(Ca) channel activity in cultured rat cerebellar Purkinje neurons was selectively inhibited by antisense oligonucleotide sequence (3 microM) targeted against the rslo transcript. The knockdown was specific; delayed rectifier and apamin-sensitive K+ channel abundances in Purkinje neurons were not affected by rslo antisense. Sense oligonucleotides (3 microM), used as a control, had no effect on channel abundance. Quantitative morphometric analyses of anti-calbindin-labeled Purkinje neurons showed no differences between neurons in control, sense and antisense treatment groups, and confirmed that the presence of the added oligonucleotide in the sense and antisense treatment conditions had no discernable toxic effects on neuronal health, for which neurite outgrowth is a sensitive indicator. These results confirm the identification of the developmentally regulated K(Ca) channel as the product of the gene rslo in cerebellar Purkinje neurons.

Differential expression of three classes of voltage-gated Ca(2+) channels during maturation of the rat cerebellum in vitro.

Voltage-gated Ca(2+) channels provide a mode of Ca(2+) influx that is essential for intracellular signaling in many cells. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the relative amounts of mRNAs encoding three classes of Ca(2+) channels (alpha1A, alpha1B and alpha1E) during development, in cultures established from prenatal rat cerebellar cortex. Ca(2+) channel transcript levels were standardized to a constitutive marker (cyclophilin). For all three classes of Ca(2+) channels, transcript levels were highest at early stages (4-10 days in vitro) and declined with age. This developmental pattern was differentially regulated by a depolarizing agent, tetraethylammonium chloride (TEA, 1 mM). Chronic depolarization yielded a significant elevation in transcript levels for alpha1B (N-type) and alpha1E (R-type) Ca(2+) channels during neuronal maturation (10-21 days in vitro), but dramatically suppressed transcript levels for the alpha1A (P-type) Ca(2+) channel at all stages of development. The effects of TEA on alpha1A, alpha1B and alpha1E transcript levels were mimicked by increasing external K(+) (from 5 to 10 mM). The regulatory effects of depolarization on transcript levels were dependent on extracellular Ca(2+) for alpha1E but not for alpha1A. For alpha1B, transcript levels depended on extracellular Ca(2+) only for increased K(+) as the depolarizing stimulus, but not for TEA. These results suggest that levels of Ca(2+) channel transcripts in rat cerebellum are developmentally regulated in vitro and can be influenced differentially by transmembrane signaling via chronic depolarization and Ca(2+) entry. Dynamic regulation of Ca(2+) channel expression may be relevant to the different functional roles of Ca(2+) channels and their regional localization within neurons.

Multiple voltage-sensitive K+ channels regulate dendritic excitability in cerebellar Purkinje neurons.

Ionic conductances present in the dendritic region of the cerebellar Purkinje neuron were studied using the single-channel and whole-cell recording methods. Several types of voltage-sensitive K+ channels including a Ca2+ activated K+ channel were found to be a prominent components of the dendritic membrane. All patches studied contained K+ channel types and most patches contained more than one K+ channel type. In cell attached recordings, K+ channel activity was associated with the late phase of spontaneous action potentials suggesting a functional relationship. These data demonstrate that voltage-sensitive ion channels contribute to dendritic excitability and suggest that the transduction and integration of synaptic signals may involve both active and passive ionic conductances.

Fluid-percussion brain injury induces changes in aquaporin channel expression.

Edema, the accumulation of excess fluid, is a major pathological change in the brain that contributes significantly to pathology and mortality after moderate to severe brain injury. Edema is regulated by aquaporin (AQP) channels which transport water across cellular membranes. Six AQPs are found in the brain (1, 3, 4, 5, 8, and 9), and previous studies have found that AQP4 is regulated after traumatic brain injury (TBI). To further understand how AQPs contribute to brain edema, we investigated whether expression of AQP1, 3, and 9 are also regulated after TBI. Adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury (FPI) or sham surgery. After induction of FPI, the injured, ipsilateral parietal cortex and hippocampus were dissected and analyzed by Western blotting. We observed a small decrease in AQP3 and 4 levels at 7 days after FPI in the ipsilateral, parietal cortex. Both AQP1 and 9 significantly increased within 30 min post-injury and remained elevated for up to 6 h in the ipsilateral, parietal cortex. Aqp1 and 9 mRNA levels were also significantly increased at 30 min post-FPI. Administration of an AQP1 and 4 antagonist, AqB013, non-significantly increased brain water content in sham, non-injured animals, and did not prevent edema formation 24 h after trauma in either the parietal cortex or hippocampus. These results indicate that Aqp1 and 9 mRNA and protein levels increase after moderate parasagittal FPI and that an inhibitor of AQP1 and 4 does not decrease edema after moderate parasagittal FPI.

Ocean fertilization: a potential means of geoengineering?

The oceans sequester carbon from the atmosphere partly as a result of biological productivity. Over much of the ocean surface, this productivity is limited by essential nutrients and we discuss whether it is likely that sequestration can be enhanced by supplying limiting nutrients. Various methods of supply have been suggested and we discuss the efficacy of each and the potential side effects that may develop as a result. Our conclusion is that these methods have the potential to enhance sequestration but that the current level of knowledge from the observations and modelling carried out to date does not provide a sound foundation on which to make clear predictions or recommendations. For ocean fertilization to become a viable option to sequester CO2, we need more extensive and targeted fieldwork and better mathematical models of ocean biogeochemical processes. Models are needed both to interpret field observations and to make reliable predictions about the side effects of large-scale fertilization. They would also be an essential tool with which to verify that sequestration has effectively taken place. There is considerable urgency to address climate change mitigation and this demands that new fieldwork plans are developed rapidly. In contrast to previous experiments, these must focus on the specific objective which is to assess the possibilities of CO2 sequestration through fertilization.

Block of the inactivating potassium channel by clofilium and hydroxylamine depends on the sequence of the pore region.

Cardiac antiarrhythmic compounds are a diverse group divided into classes that differ in their mechanisms of action. Recent attention has focused on class III compounds, which prolong the action potential by blocking K+ channels. The purpose of this study was to characterize the mechanisms of actions of a class III compound, clofilium, and a simple analog, hydroxylamine, on an inactivating K+ channel. The defined system used a cloned inactivating K+ channel (Shaker-B) expressed in Xenopus oocytes. This channel is similar in physiological properties and core sequence to the inactivating K+ channel cloned from mammalian heart. Results presented here demonstrate that clofilium (100 microM) and hydroxylamine (10 mM) can cause use-dependent block, depending on the sequence of the pore region. A mutation of the pore known to influence selectivity and tetraethylammonium binding (threonine-441 to serine) confers use-dependent sensitivity to hydroxylamine and clofilium. Hybrid channels were formed from the coinjection of wild-type and mutant channel mRNAs; the analysis of block with the hybrid channels suggests that binding of hydroxylamine involves all subunits of the tetrameric channel, whereas clofilium affects channels containing as few as one mutant subunit. The simplest interpretation is that all four subunits contribute to an internal binding site for blockers such as clofilium and hydroxylamine and threonine-441 influences this binding site. The effectiveness of clofilium, unlike hydroxylamine, on the hybrid channels may reflect its structural complexity, which could allow interaction with a broader receptor site. Future studies will test this idea using other class III-related compounds.

Alteration of ionic selectivity of a K+ channel by mutation of the H5 region.

The high ionic selectivity of K+ channels is a unifying feature of this diverse class of membrane proteins. Though K+ channels differ widely in regulation and kinetics, physiological studies have suggested a common structure: a single file pore containing multiple ion-binding sites and having broader vestibules at both ends. We have used site-directed mutagenesis and single-channel recordings to identify a molecular region that influences ionic selectivity in a cloned A-type K+ channel from Drosophila. Single amino-acid substitutions in H5, the fifth hydrophobic region, enhanced the passage of NH4+ and Rb+, ions with diameters larger than K+, without compromising the ability of the channel to exclude the smaller cation, Na+. The mutations that substantially altered selectivity had little effect on the gating properties of the channel. We conclude that the H5 region is likely to line the pore of the K+ channel.