Cells maintain a negative resting membrane potential through the constitutive activity of background K+ channels. A novel multigene family of such K+ channels has recently been identified. A unique characteristic of these K+ channels is the presence of two homologous, subunit‐like domains, each containing a pore‐forming region. Sequence co‐variations in the GYGD signature motifs of the two pore regions suggested an interaction between neighbouring pore domains. Mutations of the GYGD motif in the rat drk1 (Kv2.1) K+ channel showed that the tyrosine (Y) position was important for K+ selectivity and single channel conductance, whereas the aspartate (D) position was a critical determinant of open state stability. Tandem constructs engineered to mimic the GYGx‐GxGD pattern seen in two‐domain K+ channels delineated a co‐operative intersubunit interaction between the Y and D positions, which determined ion selectivity, conductance and gating. In the bacterial KcsA K+ channel crystal structure, the equivalent aspartate residue (D80) does not directly interact with permeating K+ ions. However, the data presented here show that the D position is able to fine‐tune ion selectivity through a functional interaction with the Y position in the neighbouring subunit. These data indicate a physiological basis for the extensive sequence variation seen in the GYGD motifs of two‐domain K+ channels. It is suggested that a cell can precisely regulate its resting membrane potential by selectively expressing a complement of two‐domain K+ channels.
The pore of potassium channels is lined by four identical, highly conserved hairpin loops, symmetrically arranged around a central permeation pathway. Introduction of cysteines into the external mouth of the drk1 K channel pore resulted in the formation of disulfide bonds that were incompatible with channel function. Breaking these bonds restored function and resulted in a high-affinity Cd(2+)-binding site, indicating coordinated ligation by multiple sulfhydryls. Dimeric constructs showed that these disulfide bonds formed between subunits. These results impose narrow constraints on intersubunit atomic distances in the pore that strongly support a radial pore model. The data also suggest an important functional role for the outer mouth of the pore in gating or permeation.
The calcium channel ␣ 1A subunit gene codes for proteins with diverse structure and function. This diversity may be important for fine tuning neurotransmitter release at central and peripheral synapses. The ␣ 1A C terminus, which serves a critical role in processing information from intracellular signaling molecules, is capable of undergoing extensive alternative splicing. The purpose of this study was to determine the extent to which C-terminal alternative splicing affects some of the fundamental biophysical properties of ␣ 1A subunits. Specifically, the biophysical properties of two alternatively spliced ␣ 1A subunits were compared. One variant was identical to an isoform identified previously in human brain, and the other was a novel isoform isolated from human spinal cord. The variants differed by two amino acids (NP) in the extracellular linker between transmembrane segments IVS3 and IVS4 and in two C-terminal regions encoded by exons 37 and 44. Expression in Xenopus oocytes demonstrated that the two variants were similar with respect to current-voltage relationships and the voltage dependence of steady-state activation and inactivation. However, the rates of activation, inactivation, deactivation, and recovery from inactivation were all significantly slower for the spinal cord variant. A chimeric strategy demonstrated that the inclusion of the sequence encoded by exon 44 specifically affects the rate of inactivation. These findings demonstrate that C-terminal structural changes alone can influence the way in which ␣ 1A subunits respond to a depolarizing stimulus and add to the developing picture of the C terminus as a critical domain in the regulation of Ca 2ϩ channel function.
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