The implementation of clay modeling and rat dissection into the human anatomy and physiology curriculum of a large urban community college.

After a considerable amount of research and experimentation, cat dissection was replaced with rat dissection and clay modeling in the human anatomy and physiology laboratory curricula at La Guardia Community College (LAGCC), a large urban community college of the City University of New York (CUNY). This article describes the challenges faculty overcame and the techniques used to solve them. Methods involved were: developing a laboratory manual in conjunction with the publisher, holding training sessions for faculty and staff, the development of instructional outlines for students and lesson plans for faculty, the installation of storage facilities to hold mannequins instead of cat specimens, and designing mannequin clean-up techniques that could be used by more than one thousand students each semester. The effectiveness of these curricular changes was assessed by examining student muscle practical examination grades and the responses of faculty and students to questionnaires. The results demonstrated that the majority of faculty felt prepared to teach using clay modeling and believed the activity was effective in presenting lesson content. Students undertaking clay modeling had significantly higher muscle practical examination grades than students undertaking cat dissection, and the majority of students believed that clay modeling was an effective technique to learn human skeletal, respiratory, and cardiovascular anatomy, which included the names and locations of blood vessels. Furthermore, the majority of students felt that rat dissection helped them learn nervous, digestive, urinary, and reproductive system anatomy. Faculty experience at LAGCC may serve as a resource to other academic institutions developing new curricula for large, on-going courses.

Positions of ß2 and ß3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel.

Large-conductance voltage- and Ca(2+)-gated K(+) channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory ß subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ∼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in ß2 and ß3, as in ß1 and ß4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse ß3 (mß3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0-αS6. We identified by elimination mß3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in ß1. These are conserved in ß2-ß4, which have four additional Cys (eight in total), except that mß3 has one fewer. In ß1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In mß3, the free Cys is at position 7; position 2 lacks a Cys present in all other ß2-ß4; and the disulfide pattern is 1-8, 3-4, and 5-6. Presumably, Cys 2 cross-links to Cys 7 in all other ß2-ß4. Cross-linking of mß3 Cys152 to Cys substituted in the flanks of αS0-S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the αS6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of mß3 perturbed. Although the mß3 loop can move, its position with Cys152 near αK296, in which it blocks IbTX binding, is likely favored.

Location of modulatory beta subunits in BK potassium channels.

Large-conductance voltage- and calcium-activated potassium (BK) channels contain four pore-forming alpha subunits and four modulatory beta subunits. From the extents of disulfide cross-linking in channels on the cell surface between cysteine (Cys) substituted for residues in the first turns in the membrane of the S0 transmembrane (TM) helix, unique to BK alpha, and of the voltage-sensing domain TM helices S1-S4, we infer that S0 is next to S3 and S4, but not to S1 and S2. Furthermore, of the two beta1 TM helices, TM2 is next to S0, and TM1 is next to TM2. Coexpression of alpha with two substituted Cys's, one in S0 and one in S2, and beta1 also with two substituted Cys's, one in TM1 and one in TM2, resulted in two alphas cross-linked by one beta. Thus, each beta lies between and can interact with the voltage-sensing domains of two adjacent alpha subunits.

Location of the beta 4 transmembrane helices in the BK potassium channel.

Large-conductance, voltage- and Ca(2+)-gated potassium (BK) channels control excitability in a number of cell types. BK channels are composed of alpha subunits, which contain the voltage-sensor domains and the Ca(2+)- sensor domains and form the pore, and often one of four types of beta subunits, which modulate the channel in a cell-specific manner. beta 4 is expressed in neurons throughout the brain. Deletion of beta 4 in mice causes temporal lobe epilepsy. Compared with channels composed of alpha alone, channels composed of alpha and beta 4 activate and deactivate more slowly. We inferred the locations of the two beta 4 transmembrane (TM) helices TM1 and TM2 relative to the seven alpha TM helices, S0-S6, from the extent of disulfide bond formation between cysteines substituted in the extracellular flanks of these TM helices. We found that beta 4 TM2 is close to alpha S0 and that beta 4 TM1 is close to both alpha S1 and S2. At least at their extracellular ends, TM1 and TM2 are not close to S3-S6. In six of eight of the most highly crosslinked cysteine pairs, four crosslinks from TM2 to S0 and one each from TM1 to S1 and S2 had small effects on the V(50) and on the rates of activation and deactivation. That disulfide crosslinking caused only small functional perturbations is consistent with the proximity of the extracellular ends of TM2 to S0 and of TM1 to S1 and to S2, in both the open and closed states.

Clay modeling as a method to learn human muscles: A community college study.

The efficacy of clay modeling compared with cat dissection for human muscle identification was examined over two semesters at LaGuardia Community College in Queens, NY. The 181 students in 10 sections in this study were randomly distributed into control (cat dissection) and experimental (clay modeling) groups, and the results of the muscle practical examination were analyzed. The clay-modeling group was significantly better at identifying human muscles on human models than the cat-dissection group, and was as good at identifying muscles on their self-made clay mannequins as the cat-dissection group was at identifying cat muscle on their specimens. This study demonstrated that clay modeling is more effective than cat dissection for learning human muscles at the community college level.

Expression of human ERG K+ channels in the mouse heart exerts anti-arrhythmic activity.

OBJECTIVE: The K(+) channel encoded by the human ether-a-go-go-related gene (HERG) is crucial for repolarization in the human heart. In order to investigate the impact of HERG current (I(Kr)) on the incidence of cardiac arrhythmias, we generated a transgenic mouse expressing HERG specifically in the heart. METHODS AND RESULTS: ECG recordings at baseline showed no obvious difference between transgenic and wild-type (WT) mice with the exception of the T wave, which was more negative in transgenic mice than in WT mice. E4031 (20 mg/kg) prolonged the QTc interval and flattened the T wave in transgenic mice, but not in WT mice. Injection of BaCl(2) (25 mg/kg) induced short runs of ventricular tachycardia in 9/10 WT mice, but not in transgenic animals. Atrial pacing reproducibly induced atrial tachyarrhythmias in 11/15 WT mice. In contrast, atrial arrhythmia was inducible in only 2/11 transgenic mice. When pretreated with dofetilide (10 mg/kg), transgenic mice were as sensitive to experimental arrhythmias as WT mice. Microelectrode studies showed that atrial action potentials have a steeper slope of duration-rate adaptation in WT than in transgenic mice. Transgenic mice were also characterized by a post-repolarization refractoriness, which could result from the substantial amount of I(Kr) subsisting after repolarization as assessed with action potential-clamp experiments and simulations with a model of the transgenic mouse action potential. CONCLUSION: HERG expression in the mouse heart can protect against experimental induction of arrhythmias. This is the first report of such a protective effect of HERG in vivo.

Regulatory actions of the A-kinase anchoring protein Yotiao on a heart potassium channel downstream of PKA phosphorylation.

A-kinase anchoring proteins (AKAPs) are thought to be passive members of protein complexes that coordinate the association of cAMP-dependent protein kinase A (PKA) with cellular substrates to facilitate targeted PKA protein phosphorylation. I(Ks), the slow heart potassium current, is carried by the I(Ks) potassium channel, a substrate for PKA phosphorylation in response to sympathetic nerve stimulation, is a macromolecular complex that includes the KCNQ1 alpha subunit, the KCNE1 regulatory subunit, and the AKAP Yotiao. Disruption of this regulation by mutation in the long QT syndrome is associated with elevated risk of sudden death. Here, we have studied the effects of the AKAP Yotiao on the function of the I(Ks) channel that had been mutated to simulate channel phosphorylation, and we report direct AKAP-mediated alteration of channel function distinct from its role in the coordination of channel phosphorylation by PKA. These data reveal previously undescribed actions of Yotiao that occur subsequent to channel phosphorylation and provide evidence that this adaptor protein also may serve as an effector in regulating this important ion channel.

The Na+ channel inactivation gate is a molecular complex: a novel role of the COOH-terminal domain.

Electrical activity in nerve, skeletal muscle, and heart requires finely tuned activity of voltage-gated Na+ channels that open and then enter a nonconducting inactivated state upon depolarization. Inactivation occurs when the gate, the cytoplasmic loop linking domains III and IV of the alpha subunit, occludes the open pore. Subtle destabilization of inactivation by mutation is causally associated with diverse human disease. Here we show for the first time that the inactivation gate is a molecular complex consisting of the III-IV loop and the COOH terminus (C-T), which is necessary to stabilize the closed gate and minimize channel reopening. When this interaction is disrupted by mutation, inactivation is destabilized allowing a small, but important, fraction of channels to reopen, conduct inward current, and delay cellular repolarization. Thus, our results demonstrate for the first time that physiologically crucial stabilization of inactivation of the Na+ channel requires complex interactions of intracellular structures and indicate a novel structural role of the C-T domain in this process.

Requirement of a macromolecular signaling complex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel.

Sympathetic nervous system (SNS) regulation of cardiac action potential duration (APD) is mediated by beta adrenergic receptor (betaAR) activation, which increases the slow outward potassium ion current (IKS). Mutations in two human I(KS) channel subunits, hKCNQ1 and hKCNE1, prolong APD and cause inherited cardiac arrhythmias known as LQTS (long QT syndrome). We show that betaAR modulation of I(KS) requires targeting of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) and protein phosphatase 1 (PP1) to hKCNQ1 through the targeting protein yotiao. Yotiao binds to hKCNQ1 by a leucine zipper motif, which is disrupted by an LQTS mutation (hKCNQ1-G589D). Identification of the hKCNQ1 macromolecular complex provides a mechanism for SNS modulation of cardiac APD through IKS.