Functional calcium channels present in purified skeletal muscle transverse tubules were inserted into planar phospholipid bilayers composed of the neutral lipid phosphatidylethanolamine (PE), the negatively charged lipid phosphatidylserine (PS), and mixtures of both. The lengthening of the mean open time and stabilization ofsingle channel fluctuations under constant holding potentials was accomplished by the use of the agonist Bay K8644. It was found that the barium current carried through the channel saturates as a function of the BaCl2 concentration at a maximum current of 0.6 pA (at a holding potential of 0 mV) and a half-saturation value of 40 mM . Under saturation, the slope conductance of the channel is 20 pS at voltages more negative than -50 mV and 13 pS at a holding potential of 0 mV. At barium concentrations above and below the half-saturation point, the open channel currents were independent of the bilayer mole fraction of PS from Xps = 0 (pure PE) to Xrs = 1 .0 (pure PS) . It is shown that in the absence of barium, the calcium channel transports sodium or potassium ions (P N,/P K = 1 .4) at saturating rates higher than those for barium alone . The sodium conductance in pure PE bilayers saturates as a function of NaCl concentration, following a curve that can be described as a rectangular hyperbola with a half-saturation value of 200 mM and a maximum conductance of 68 pS (slope conductance at a holding potential of 0 mV). In pure PS bilayers, the sodium conductance is about twice that measured in PE at concentrations below 100 mM NaCl. The maximum channel conductance at high ionic strength is unaffected by the lipid charge . This effect at low ionic strength was analyzed according to J. Bell and C. Miller (1984 . Biophysical ,Journal . 45 :279-287) and interpreted as if the conduction pathway of the calcium channel were separated from the bilayer lipid by^-20 A. This distance thereby effectively insulates the ion entry to the channel from the bulk of the bilayer lipid surface charge . Current vs. voltage curves measured in NaCl in pure PE and pure PS show that similarly small surface charge effects are

The recently described calcium channel agonists Bay-K8644 and CGP-28392 have been used to induce long-term opening of calcium channels from purified rat muscle transverse tubules (t-tubules) incorporated into planar phospholipid bilayers. Agonist-open channels are selective for divalent cations (except Mg++), display voltage-dependent kinetics, and are blocked by the calcium channel antagonist, nitrendipine. The sensitivity to dihydropyridine agonists and antagonists indicate that a pool of t-tubule calcium channels remain functional after membrane fractionation and purification.

The EPC-9 computer-controlled amplifier has no front-panel controls; therefore the user interface to the EPC-9 patch-clamp amplifier is defined entirely by software. This paper describes various user interfaces that have been implemented, including a high-level programming interface, a user interface based on the PostScript language, and graphical user interfaces that control the EPC-9 from data-acquisition programs. Also described are the algorithms used for automatic adjustment of the C-Fast and C-Slow transient cancellation circuitry. An overview of the procedures that perform automatic testing and calibration is given.

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The verapamil-type calcium antagonist, D600, and its charged quaternary derivative, D890, were used to assess the sidedness of blockade in single calcium channels reconstituted from purified transverse tubules of skeletal muscle. Spontaneous single channel openings were induced with the agonist Bay-K8644 and recordings were made in a two-chamber planar bilayer setup so that drugs could be delivered to either side of the channel. Micromolar drug addition resulted in a greater than 10-fold decrease in probability of open channel events (po) without a significant change in single channel currents. Changes in po occurred in parallel with changes in mean open time and both parameters could be titrated with a similar IC50. At pH 7.2, cis or trans D600 blocked with an IC50 of 5 microM but for D890 the IC50 was cis 3 microM and trans greater than 75 microM (cis is the intracellular-equivalent side as defined by the voltage-dependent activation). The asymmetry of D890 blockade indicates that the drug can readily gain access to the blocking site from the aqueous phase adjacent to the inner but not extracellular end of the channel.