We propose to study the molecular mechanisms underlying ionic permeability and gating of C1 and K ion channels. The experiments use embryonic or adult rat central neurons (hippocampus, spinal cord) maintained in primary tissue culture, or obtained by acute dissociation. The patch clamp technique will be used to record macroscopic membrane currents from membrane spheres excised from the cell soma, and to study currents from individual channels in small membrane patches. We will analyze the mechanism of ionic selectivity and permeability of two varieties of C1 channel: C1 channels open in the resting membrane and GABA- or glycine- activated C1 channels. Cation/anion interactions inside the resting C1 channel will be investigated to test a mechanism of joint anion/cation permeation proposed from previous work. The block by Zn ion, and effects of pH will be studied. The ionic selectivity of the transmitter-activated C1 channels will be examined and compared to that of resting C1 channels, to determine whether or not these small-conductance C1 channels achieve ionic selectivity by the same basic mechanism. Molecular channels underlying the rapidly and slowly inactivating components of voltage-activated K current will be identified. We will further study the gating of these channels, assess their role in shaping the somatic excitability, and examine their differential sensitivity to K channel blockers. We will perform kinetic measurements on single-channel gating at subzero temperatures. The behavior of the large Ca-activated K channel will be characterized for temperatures down to -30 degrees C. This new method is expected to greatly enhance the range of kinetic studies possible with ion channels.
