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R MacKinnon

Publications and source records attributed to R MacKinnon.

65 records · Page 4Linked to original sources
PubMed1989

Mutant potassium channels with altered binding of charybdotoxin, a pore-blocking peptide inhibitor.

The inhibition by charybdotoxin of A-type potassium channels expressed in Xenopus oocytes was studied for several splicing variants of the Drosophila Shaker gene and for several site-directed mutants of this channel. Charybdotoxin blocking affinity is lowered by a factor of 3.5 upon replacing glutamate-422 with glutamine, and by a factor of about 12 upon substituting lysine in this position. Replacement of glutamate-422 by aspartate had no effect on toxin affinity. Thus, the glutamate residue at position 422 of this potassium channel is near or in the externally facing mouth of the potassium conduction pathway, and the positively charged toxin is electrostatically focused toward its blocking site by the negative potential set up by glutamate-422.

Animals↗
PubMed1988

Charybdotoxin block of Shaker K+ channels suggests that different types of K+ channels share common structural features.

Charybdotoxin (CTX), a 37 amino acid protein isolated from the venom of L. quinquestriatus, is a high-affinity blocker of various Ca2(+)-activated K+ channels. CTX also blocks Drosophila Shaker (Sh) clone H4 transient K+ currents expressed in Xenopus oocytes with similar affinity (Kd = 3.6 nM). CTX blocks both the open and the closed states of Sh channels with no apparent change in gating behavior. In addition, the block is enhanced as the ionic strength is lowered. These properties are identical to those of CTX block of Ca(+)-activated K+ channels, and these results suggest that the external pore openings of these two functionally dissimilar K+ channels may share common structural features.

Animals↗
PubMed1988

Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength.

Charybdotoxin (CTX), a small, basic protein from scorpion venom, strongly inhibits the conduction of K ions through high-conductance, Ca2+-activated K+ channels. The interaction of CTX with Ca2+-activated K+ channels from rat skeletal muscle plasma membranes was studied by inserting single channels into uncharged planar phospholipid bilayers. CTX blocks K+ conduction by binding to the external side of the channel, with an apparent dissociation constant of approximately 10 nM at physiological ionic strength. The dwell-time distributions of both blocked and unblocked states are single-exponential. The toxin association rate varies linearly with the CTX concentration, and the dissociation rate is independent of it. CTX is competent to block both open and closed channels; the association rate is sevenfold faster for the open channel, while the dissociation rate is the same for both channel conformations. Membrane depolarization enhances the CTX dissociation rate e-fold/28 mV; if the channel's open probability is maintained constant as voltage varies, then the toxin association rate is voltage independent. Increasing the external solution ionic strength from 20 to 300 mM (with K+, Na+, or arginine+) reduces the association rate by two orders of magnitude, with little effect on the dissociation rate. We conclude that CTX binding to the Ca2+-activated K+ channel is a bimolecular process, and that the CTX interaction senses both voltage and the channel's conformational state. We further propose that a region of fixed negative charge exists near the channel's CTX-binding site.

Animals↗
PubMed1988

Mechanism of charybdotoxin block of the high-conductance, Ca2+-activated K+ channel.

The mechanism of charybdotoxin (CTX) block of single Ca2+-activated K+ channels from rat muscle was studied in planar lipid bilayers. CTX blocks the channel from the external solution, and K+ in the internal solution specifically relieves toxin block. The effect of K+ is due solely to an enhancement of the CTX dissociation rate. As internal K+ is raised, the CTX dissociation rate increases in a rectangular hyperbolic fashion from a minimum value at low K+ of 0.01 s-1 to a maximum value of approximately 0.2 s-1. As the membrane is depolarized, internal K+ more effectively accelerates CTX dissociation. As the membrane is hyperpolarized, the toxin dissociation rate approaches 0.01 s-1, regardless of the K+ concentration. When internal K+ is replaced by Na+, CTX dissociation is no longer voltage dependent. The permeant ion Rb also accelerates toxin dissociation from the internal solution, while the impermeant ions Li, Na, Cs, and arginine do not. These results argue that K ions can enter the CTX-blocked channel from the internal solution to reach a site located nearly all the way through the conduction pathway; when K+ occupies this site, CTX is destabilized on its blocking site by approximately 1.8 kcal/mol. The most natural way to accommodate these conclusions is to assume that CTX physically plugs the channel's externally facing mouth.

Animals↗
PubMed1988

Modulation by the thyroid state of intracellular calcium and contractility in ferret ventricular muscle.

The purpose of this study was to determine whether the cardiac contractile abnormalities induced by hyperthyroidism or hypothyroidism are caused by changes in intracellular Ca2+ handling or by alterations in the distribution of isoenzymes of ventricular myosin. Right ventricular papillary muscles obtained from euthyroid ferrets and ferrets treated with L-thyroxine (hyperthyroid) or methimazole (hypothyroid) were loaded with the calcium indicator aequorin for recording intracellular Ca2+ levels during isometric contraction. In muscles from the hypothyroid ferrets, peak tension was reduced and the duration of contraction prolonged compared to the controls; these changes were associated with a Ca2+ transient of decreased amplitude and prolonged duration. Hyperthyroidism produced opposite changes in the time course of the Ca2+ transient and the associated isometric contraction. Native polyacrylamide gel electrophoresis was performed on myosin extracted from the right ventricular free wall of control and treated ferrets. The hyperthyroid state was associated with new myosin formation as indicated by the appearance of three myosin bands on the pyrophosphate gel. Gels of myosin from hypothyroid and euthyroid ferrets showed a single band that migrated with the slowest of the three bands from the hyperthyroid ferrets. These results suggest that changes in both Ca2+ handling and myosin isoenzymes may contribute to the contractile abnormalities observed in hyperthyroidism. Alterations in intracellular Ca2+ handling alone may account for the contractile changes induced by hypothyroidism.

Animals↗
PubMed1987

Differential effects of reoxygenation on intracellular calcium and isometric tension.

We used the bioluminescent Ca2+ indicator, aequorin to record intracellular calcium transients during reoxygenation of hypoxic ferret ventricular muscle in order to determine whether alterations in the amplitude and time course of isometric contraction are mediated by changes in [Ca2+]i. Papillary muscles less than or equal to 1 mm in diameter were removed from the hearts of male ferrets and perfused with a bicarbonate-buffered physiologic salt solution at 30 degrees C. Muscles were stimulated to contract isometrically at 0.33 Hz and were loaded with aequorin by a chemical procedure. Hypoxia was induced by changing the gas mixture bubbling the perfusate to 95% N2, 5% CO2; reoxygenation was accomplished by switching the gas mixture to 95% O2, 5% CO2. Hypoxia produced a decrease in peak Ca2+ and tension that was reversed by reoxygenation. However, the effects on tension of changes in oxygenation were greater than expected from the degree of change in [Ca2+]i. The time courses of the Ca2+ transient and isometric twitch moved in opposite directions and were respectively prolonged/abbreviated by hypoxia and abbreviated/prolonged by reoxygenation. These results indicate that changes in the amplitude and time course of the isometric twitch induced by hypoxia and reoxygenation cannot be attributed alone to changes in intracellular Ca2+ availability and are caused in part by a significant decrease in the calcium sensitivity of the contractile apparatus.

Aequorin↗
PubMed1987

Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure.

Intracellular Ca2+ release and reuptake are essential for contraction and relaxation of normal heart muscle. Intracellular Ca2+ transients were recorded with aequorin during isometric contraction of myocardium from patients with end-stage heart failure. In contrast to controls, contractions and Ca2+ transients of muscles from failing hearts were markedly prolonged, and the Ca2+ transients exhibited 2 distinct components. Muscles from failing hearts showed a diminished capacity to restore low resting Ca2+ levels during diastole. These experiments provide the first direct evidence from actively contracting human myocardium that intracellular Ca2+ handling is abnormal and may cause systolic and diastolic dysfunction in heart failure.

Adolescent↗
PubMed1986

Influence of the thyroid state on the calcium transient in ventricular muscle.

The purpose of this study was to determine the influence of thyroid hormone on tension development and the intracellular calcium transient in mammalian ventricular muscle. A hyperthyroid (H) state was induced in ferrets by subcutaneous injection of L-thyroxine, 0.3 mg/kg daily, for 2-3 weeks. One-half of the age matched control group (C) were injected with vehicle. Aequorin was loaded into the cells of ferret papillary muscles by a chemical procedure. The muscles were stimulated at 0.33 Hz and isometric tension and the calcium transient were simultaneously recorded at 30 degrees C. Peak isometric tension in mN/mm2 (+/- SD) was 15.4 +/- 7.2 and 16.2 +/- 7.9 for C (n = 8) and H (n = 9) respectively. The time to peak tension and time to 80% relaxation from peak of tension were reduced by 22% and 28% respectively in H compared to C. After stimulation, the calcium transient reached a maximum in 56 +/- 6 ms in C and in 47 +/- 5 ms in H. The time to 80% decay of the peak calcium transient was 95 +/- 8 ms and 68 +/- 5 ms for C and H respectively. The ratio of the aequorin luminescence at the peak of the calcium transient over the calculated maximum luminescence, Lmax, were compared and they were not different. At 22 degrees C Log (L/Lmax) was -3.3 +/- 0.1 in C (n = 4) and -3.4 +/- 0.3 in H (n = 3). These results indicate that the thyroid state influences the time course of the calcium transient and are consistent with the abbreviation in the duration of contraction that is observed in the hyperthyroid state.

Aequorin↗
PubMed1980

Purification of human alpha uterine protein.

Human alpha uterine protein (AUP) has been prepared from extracts of decudua by antibody affinity chromatography, DEAE Sepharose chromatography and by filtration through Sephadex G-150. This procedure yielded a protein fraction containing AUP, which was labelled with 125I by chloramine T. When analysed by SDS gel electrophoresis this radioiodinated protein fraction was found to contain predominantly a single species of protein which was precipitated by antibodies against AUP in antibody-antigen crossed electrophoresis. Rabbit anti-AUP precipitated 55-65% of the tracer in a double-antibody system. Sephadex G150 gel filtration of AUP obtained before and after affinity chromatography provided a molecular weight estimate of 50000. Since SDS gel electrophoresis revealed a polypeptide molecular weight of 23000-25000, it is suggested that AUP is a dimer.

Chromatography, Affinity↗
PubMed1975

A clinical clerkship in psychiatry.

A clinical clerkship was organized around the goal of teaching information and skills that would be needed by the nonpsychiatrist physician. In most clinical clerkships the students work on an inpatient service. In the clerkship described here the setting is an outpatient clinic, which provides a more relevant experience. Videotaped psychiatric interviews are used extensively and have been found to provide a valuable teaching format. They are effective in holding student interest, in avoiding the practical difficulties of live interviews, and in teaching active listening and interviewing technique, as well as in demonstrating a variety of psychopathology. Field trips to state psychiatric hospitals, institutions for mental defectives, clinics for treating alcoholics or addicts, and other mental health facilities have been used but have not been found to be a very valuable part of the program.

Education, Medical, Undergraduate↗