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A B S T R A C T The effects of the two local anesthetics tetracaine and procaine and a quaternary amine derivative of lidocaine, QX314, on sarcoplasmic reticulum (SR) Ca 2+ release have been examined by incorporating the purified rabbit skeletal muscle Ca 2+ release channel complex into planar lipid bilayers. Recordings of potassium ion currents through single channels showed that Ca 2÷-and ATP-gated channel activity was reduced by the addition of the tertiary amines tetracaine and procaine to the c/s (cytoplasmic side of SR membrane) or tram (SR lumenal) side of the bilayer. Channel open probability was lowered twofold at tetracaine and procaine concentrations of ~ 150 tzM and 4 raM, respectively. Hill coefficients of 2.0 and greater indicated that the two drugs inhibited channel activity by binding to two or more cooperatively interacting sites. Unitary conductance of the K +-conducting channel was not changed by 1 mM tetracaine in the c/s and trans chambers. In contrast, c/s millimolar concentrations of the quaternary amine QX314 induced a fast blocking effect at positive holding potentials without an apparent change in channel open probability. A voltage-dependent block was observed at high concentrations (millimolar) of tetracaine, procaine, and QX314 in the presence of 2 ~M ryanodine which induced the formation of a long open subconductance. Vesicle-4~Ca 2+ ion flux measurements also indicated an inhibition of the SR Ca 9+ release channel by tetracaine and procaine. These results indicate that local anesthetics bind to two or more cooperatively interacting high-affinity regulatory sites of the Ca 2+ release channel in or close to the SR membrane. Voltagedependent blockade of the channel by QX314 in the absence of ryanodine, and by QX314, procaine and tetracaine in the presence of ryanodine, indicated one low-affinity site within the conduction pathway of the channel. Our results further suggest that tetracaine and procaine may primarily inhibit excitation-contraction coupling in skeletal muscle by binding to the high-affinity, regulatory sites of the SR Ca 2+ release channel.

We discuss a technique to compute (K) using templates developed by fitting a Fourier series to existing high-quality K light curves of field RR Lyraes. We find that a series of order 2 is sufficient to model the light curves of first-overtone RRc variables, but four different sixth-order templates are needed for the fundamental RRab stars due to changes in the light curves that appear to correlate with the B amplitude. Applying the appropriate template to single-phase observations yield estimated (K) values whose deviation from the true (K) is randomly distributed over phase, and is of the same order of magnitude as the observational uncertainty, as long as the ephemeris phase is accurate. The addition of a second point, separated by at least 0.2 in phase from the first, allows the use of template shifting to remove deviations that may arise from uncertainties in the ephemeris phase, and template and scaling factor selection, with final systematic errors reduced to less than 0.03 mag. We find that the use of templates yield superior results to those derived using other techniques, which can produce (K) values that show systematic deviations over phase.

The CHAPS-solubilized and purified 30S ryanodine receptor protein complex from skeletal sarcoplasmic reticulum (SR) was incorporated into planar lipid bilayers. The resulting electrical activity displayed similar responses to agents such as Ca2+, ATP, ryanodine, or caffeine as the native Ca2+ release channel, confirming the identification of the 30S complex as the Ca2+ release channel. The purified channel was permeable to monovalent ions such as Na+, with the permeability ratio PCa/PNa approximately 5, and was highly selective for cations over anions. The purified channel also showed at least four distinct conductance levels for both Na+ and Ca2+ conducting ions, with the major subconducting level in NaCl buffers possessing half the conductance value of the main conductance state. These levels may be produced by intrinsic subconductances present within the channel oligomer. Several of these conductances may be cooperatively coupled to produce the characteristic 100 +/- 10 pS unitary Ca2+ conductance of the native channel.