Biomedical subjects

Paul Pfaffinger

Publications and source records attributed to Paul Pfaffinger.

4 recordsLinked to original sources

PubMed2005

Acceleration of K+ channel inactivation by MEK inhibitor U0126.

Voltage-dependent (Kv)4.2-encoded A-type K+ channels play an important role in controlling neuronal excitability and are subject to modulation by various protein kinases, including ERK. In studies of ERK modulation, the organic compound U0126 is often used to suppress the activity of MEK, which is a kinase immediately upstream from ERK. We have observed that the inactivation time constant of heterologously expressed Kv4.2 channels was accelerated by U0126 at 1-20 microM. This effect, however, was not Kv4 family specific, because U0126 also converted noninactivating K+ currents mediated by Kv1.1 subunits into transient ones. To determine whether U0126 exerted these effects through kinase inhibition, we tested U0125, a derivative of U0126 that is less potent in MEK inhibition. At the same concentrations, U0125 had effects similar to those of U0126 on channel inactivation. Finally, we expressed a mutant form of Kv4.2 in which three identified ERK phosphorylation sites (T602, T607, and S616) were replaced with alanines. The inactivation of K+ currents mediated by this mutant was still accelerated by U0126. Our data favor the conclusion that the increase in the rate of channel inactivation by U0126 is likely to be independent of protein kinase inhibition and instead represents a direct action on channel gating.

Amino Acid Sequence↗

PubMed2003

A modified Sindbis vector for prolonged gene expression in neurons.

Sindbis viruses have been widely used in neurobiology to express a variety of genes in cultured neurons, in cultured slices, and in vivo. They provide fast onset and high levels of expression of foreign genes, but the expression is limited to a short time window due to a shut-off of host protein synthesis. We have used a mutation in an essential gene (nsP2) of the life cycle of Sindbis, which allows the functional analysis of changes in protein expression for >/=6 days after infection. This Sindbis mutant (nsP2) was used to express enhanced green fluorescent protein (EGFP) in hippocampal neurons in culture and in vivo without any sign of toxicity, based on two-photon imaging and electrophysiology. In addition, the EGFP mutant virus can be injected in vivo to visualize spines and other details of neuronal structure. The Sindbis mutant described here provides an improved tool in neurobiology with reduced cytotoxicity and a prolonged time window of expression for novel applications in imaging and behavior. In addition, the use of this vector for the functional expression of mammalian voltage-gated ion channels in organotypic slices is demonstrated.

Animals↗

PubMed2002

Zinc mediates assembly of the T1 domain of the voltage-gated K channel 4.2.

An intermolecular Zn(2+)-binding site was identified in the structure of the T1 domain of the Shaw-type potassium channels (aKv3.1). T1 is a BTB/POZ-type domain responsible for the ordered assembly of voltage-gated potassium channels and interactions with other macromolecules. In this structure, a Zn(2+) ion was found to be coordinated between each of the four assembly interfaces of the T1 tetramer by three Cys and one His encoded in the sequence motif (HX(5)CX(20)CC) of the T1 domain. This sequence motif is conserved among all non-Shaker-type voltage-dependent potassium (Kv) channels, but not in Shaker-type channels. The presence of this conserved Zn(2+)-binding site is a primary molecular determinant that distinguishes the tetrameric assembly of non-Shaker Kv channel subunits from that of Shaker channels. We report here that tetramerization of the Shal (rKv4.2) T1 in solution requires the presence of Zn(2+), and the addition/removal of Zn(2+) reversibly switches the protein between a stable tetrameric or monomeric state. We further show that the conversion from tetramers to monomers is profoundly pH-dependent: as the solution pH gets lower, the dissociation rate increases significantly. The unfolding energy of the T1 tetramer as a measure of the conformational stability of the structure is also pH-dependent. Surprisingly, at a lower pH we observe a distinctly altered conformational state of the T1 tetramer trapped during the process of unfolding of the T1 tetramer in the presence of Zn(2+). The conformational alteration may be responsible for increased rate of dissociation at lower pH by allowing Zn(2+) to be removed more effectively by EDTA. The ability of the T1 domain to adopt stable alternative conformations may be essential to its function as a protein-protein interaction/signaling domain to modulate the ion conduction properties of intact full-length Kv channels.

Amino Acid Motifs↗

PubMed2002

Excitability is mediated by the T1 domain of the voltage-gated potassium channel.

The T1 domain of voltage-gated K+ (Kv) channel is the N-terminal cytoplasmic part of the channel preceding the transmembrane pore domain of the channel. Several crystal structures of the T1 domain show that the four T1 subunits are arranged in a rotationally symmetric tetramer. The subunit interface of the T1 domain encodes the assembly specificity of intact functional Kv channels. Along the fourfold symmetry axis of the T1 tetramer, a water-filled cavity exists. K+ ions, however, do not pass through this T1 cavity. Instead, they are believed to enter the transmembrane pore through four identical inter-subunit spaces created between the membrane-facing C-terminal side of the T1 tetramer and the inner leaflet of the membrane. Several point mutations have been introduced into the putative membrane-facing region of the T1 tetramer. These mutations led to a systematic change of the channel's voltage sensitivity. Such functional change was accompanied by a distinct structural change in the C-terminal membrane-facing side of the T1 tetramer. Interestingly, a similar structural alteration that renders the channel more excitable is also induced by the binding of a cytoplasmic protein Kv beta subunit. Within this conformationally flexible part of the T1 tetramer, non-Shaker type Kv channel subunits invariably contain one Zn2+ per subunit. With the Kv4.2 T1, we demonstrated that the tetramer can be reversibly converted to monomers by chelating zinc away from the protein. The rate of removal of Zn2+ is pH-dependent. The structural ability of the T1 tetramer to alter conformation could be an essential property to mediate and process protein protein interaction events in the cytoplasm to control excitability of intact full-length Kv channels.

Animals↗