All cloned voltage-dependent ion channels show a striking conservation of regularly spaced, positively charged amino acids in the putative transmembrane domain, termed S4. It has been suggested that the S4 is the voltage sensor. The broad, long-term objectives of this proposal are to evaluate the role of the S4 region in voltage sensing. We have cloned K channels from muscle (RMK1) and brain (RCK1) that when expressed in oocytes or cells show properties of a delayed rectifier K channel (1). Specific mutations of three S4 charged residues showed that the charges R1 and R2 participate in the charge movement while K7 does not. Yet, each one of these charges makes quantitatively very different contributions to the overall gating valence (z). R2 accounts for 20% of z and shows a purely electrostatic behavior. R1 accounts for 40% of z, an effect that cannot be explained by electrostatic interactions alone (2). Yet, the relative contribution of residues R3, R4, K5, and R6 remains unknown since specific mutations of these residues have resulted in complete loss of function.
The specific aims of this proposal are: 1) Having first identified and localized the RMK1 channel proteins (in particular the non-functional mutants) by immunofluorescence and immunoprecipitation we will embark on direct measurements of the gating charge movement by measuring gating currents; 2) We will assess the relative contribution of each K channel subunit S4 region in voltage sensing by creating tandem RMK1 constructs; 3) Effects of the specific amino acid content of S4 in voltage sensing by transplanting S4 regions from other channels into RMK1 and comparing the effects in the voltage sensing ability of the engineered molecules to those of the control donor ones; 4) Identification of acidic residues in putative transmembrane domains that may form salt links with the basic S4 residues. The experimental design and the methods used to carry out these projects include: site-directed mutagenesis; in vitro transcription and injection of mRNA into Xenopus oocytes; functional assessment by two microelectrode voltage clamping of oocytes of patch clamping of oocytes and cells. The RMK1 channel (each mutant separately) will be transfected into temperature sensitive (ts) COS cells which allow high level expression and, thus, direct measurements of gating charge movement with gating currents. The delayed rectifier channel offers a distinct advantage over rapidly inactivating currents by providing steady state measurements of current activation. Activation is very rapid within a small range of membrane potentials. The proposed experiments represent a comprehensive study to investigate the structural basis of voltage sensing. A better understanding of this phenomenon is essential given the central role that voltage gated ion channels play in excitable cell function.
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