Wolfgang Nonner - US grants

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.

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.

DESCRIPTION (provided by applicant): The long-term goal of this research is to determine the physical mechanisms of selective conduction and block in ion channels. The objective of this proposal is to analyze the frictional forces and resulting momentum transfer between ions and channel and also among ions. Friction and momentum transfer limit conductance, contribute to selectivity, are involved in determining the characteristics of current/voltage plots, and are important in channel blockade. They are dynamic phenomena that cannot be understood in terms of equilibrium properties, such as a channel's free energy landscape for ions. Friction and momentum transfer in the BK and KcsA potassium channels will be described by extending a theory of multi-component transport, first developed by Stefan and Maxwell, that is based on the conservation of mass and momentum. The extended theory will provide a means to link ionic currents to the physical properties of the channel while being constrained by known structural information. The application of the theory will allow the characteristic features of conduction, selectivity, and block to be understood in terms of basic physical mechanisms, and will also serve as a tool to determine friction parameters that are not readily available by other methods of analysis. A systematic analysis will be performed on published experimental potassium channel currents carried by several permeant ion species tested singly or in mixtures, as well as for symmetrical and asymmetrical solutions, and on K currents blocked with difference classes of blockers: Na+, Mg2+, and different sized sugars. This study is expected to provide insight into the role of friction and momentum transfer in crucial functions of potassium channels including conductance, rejection of Na ions, and block. Ion channels are found in all cells and are essential for the electrical activity of neurons and muscle and are involved in the function of kidney and intestine. The proposed work seeks to understand the physical principles and structural features that allow ion channels to select and conduct ions and to be blocked by various molecules. Such information will increase our understanding of ion channel function and should facilitate the development of therapeutic agents that act through ion channels.