SAP97 interacts with Kv1.5 in heterologous expression systems.

PDZ domain-containing proteins such as SAP97 and ZO-1 have been implicated in the targeting and clustering of ion channels. We have explored the interactions of these polypeptides with a cardiac voltage-gated potassium channel. Immunocytochemistry in cardiac myocytes revealed colocalization of SAP97 and Kv1.5, both at the intercalated disks and the lateral membranes. Transient transfection experiments in COS-7 cells revealed that SAP97 and Kv1.5 polypeptides formed perinuclear clustered complexes that could be coimmunoprecipitated. Mutation of the three COOH-terminal amino acid residues of Kv1.5 (T-D-L to A-A-A) abolished these interactions. Whereas in most COS-7 cells the SAP97-Kv1.5 complexes were retained in the ER, functional analyses in Xenopus oocytes showed that Kv1.5-encoded outward potassium currents were augmented by coexpression with SAP97. By contrast, cotransfected ZO-1 and Kv1.5 polypeptides in COS-7 cells could not be coprecipitated nor did the coinjection of ZO-1 augment the Kv1.5-encoded currents in oocytes. Collectively, our results suggest that SAP97 may play an important role in the modulation of Kv1.5 channel function in cardiac myocytes.

Characterization of mice with a combined suppression of I(to) and I(K,slow).

Cardiac-specific expression of a truncated Kv1.1 polypeptide (Kv1DN) attenuates the slow inactivating outward K(+) current (I(K,slow)), increases action potential duration (APD) and Q-T intervals, and induces spontaneous ventricular arrhythmias. Expression of the pore mutant of Kv4.2 (Kv4DN) eliminates the fast component of the transient outward current (I(to)) and prolongs APDs and Q-T intervals markedly; however, no arrhythmias are seen in Kv4DN mice, suggesting that APD and Q-T prolongation are not per se proarrhythmic. To test this hypothesis, the Kv1DN and Kv4DN lines were crossbred to produce animals (Kv1/Kv4DN) expressing both transgenes in an identical genetic background. Whole cell voltage-clamp recordings from left ventricular apex cells confirmed that in Kv1/Kv4DN left ventricular apex cells, both components (fast and slow) of I(to) and the 4-aminopyridine-sensitive component of I(K,slow) are eliminated, resulting in marked APD prolongation compared with wild-type, Kv1DN, or Kv4DN cells. Telemetric electrocardiogram monitoring (n = 10 mice/group) revealed a significant prolongation of Q-Tc and P-R intervals in Kv1/Kv4DN animals compared with Kv1DN or Kv4DN animals. Spontaneous arrhythmias were observed mainly in Kv1DN mice. Thus the attenuation of fast I(to) in addition to I(K,slow) in Kv1/Kv4DN mice causes significant prolongation of APD and Q-T intervals and attenuation of spontaneous arrhythmias.

Inducible polymorphic ventricular tachyarrhythmias in a transgenic mouse model with a long Q-T phenotype.

We created a mouse model with a prolonged Q-T interval and spontaneous arrhythmias by overexpressing the NH(2) terminus and first transmembrane segment (Kv1.1N206Tag) of a delayed rectifier potassium channel (LQT(+/-) mouse). Analyses were performed using whole cell recordings of cardiac myocytes, surface electrocardiography, and programmed electrical stimulation. Action potential duration (APD) was prolonged to the same extent and was more highly variable in myocytes derived from LQT(+/-) and LQT(+/+) mice than in myocytes derived from wild-type (WT) FVB mice. Under ketamine anesthesia, the Q-T interval of both LQT(+/+) and LQT(+/-) mice was comparably prolonged versus that of WT mice. Stimulation of the right ventricle using an intracardiac catheter induced polymorphic ventricular tachyarrhythmias in 50% of the LQT(+/-) mice and 36% of the LQT(+/+) mice, whereas polymorphic ventricular tachyarrhythmias were not inducible in WT mice. The analyses of LQT(+/-) and LQT(+/+) mice indicate that prolongation of the Q-T interval in LQT mice is associated with prolonged APD, increased dispersion of APD among cardiocytes, and inducibility of polymorphic ventricular tachycardia, providing the substrate for spontaneous arrhythmias in these animals.

Long QT and ventricular arrhythmias in transgenic mice expressing the N terminus and first transmembrane segment of a voltage-gated potassium channel.

Voltage-gated potassium channels control cardiac repolarization, and mutations of K+ channel genes recently have been shown to cause arrhythmias and sudden death in families with the congenital long QT syndrome. The precise mechanism by which the mutations lead to QT prolongation and arrhythmias is uncertain, however. We have shown previously that an N-terminal fragment including the first transmembrane segment of the rat delayed rectifier K+ channel Kv1.1 (Kv1.1N206Tag) coassembles with other K+ channels of the Kv1 subfamily in vitro, inhibits the currents encoded by Kv1.5 in a dominant-negative manner when coexpressed in Xenopus oocytes, and traps Kv1.5 polypeptide in the endoplasmic reticulum of GH3 cells. Here we report that transgenic mice overexpressing Kv1.1N206Tag in the heart have a prolonged QT interval and ventricular tachycardia. Cardiac myocytes from these mice have action potential prolongation caused by a significant reduction in the density of a rapidly activating, slowly inactivating, 4-aminopyridine sensitive outward K+ current. These changes correlate with a marked decrease in the level of Kv1.5 polypeptide. Thus, overexpression of a truncated K+ channel in the heart alters native K+ channel expression and has profound effects on cardiac excitability.

A cellular model for long QT syndrome. Trapping of heteromultimeric complexes consisting of truncated Kv1.1 potassium channel polypeptides and native Kv1.4 and Kv1.5 channels in the endoplasmic reticulum.

We demonstrated that overexpression of a cRNA encoding a truncated potassium channel polypeptide that contains the NH2 terminus and the first transmembrane segment (Kv1.1N206Tag) abolished the expression of Kv1.1 and Kv1.5 outward currents in Xenopus oocytes (Babila, T., Moscucci, A., Wang, H., Weaver, F. E. & Koren, G. (1994) Neuron 12, 615-626). Recently, we showed that expression of Kv1.1N206Tag in the heart of transgenic mice resulted in the creation of mice with prolongation of the surface electrocardiogram's QT interval (London, B., Han, X., Folco, E. & Koren, G. (1996) Biophys. J. 70, A2601). To study the dominant negative mechanism of Kv1.1N206Tag, we overexpressed it in GH3 cells, a pituitary cell line expressing Kv1. 5 and Kv1.4. RNase protection analysis comparing the steady-state levels of native Kv1.5 and Kv1.1N206Tag transcripts revealed an excess of Kv1.1N206Tag transcript. Immunoprecipitation analysis using 12CA5 monoclonal antibody detected a 25-kDa polypeptide in the transfected cells. The half-life of Kv1.1N206Tag was 2.6 h. Subcellular fractionation of cell lysates labeled with [35S]methionine revealed that Kv1.1N206Tag polypeptide is detectable in the particulate (membranous) fraction, but not in the soluble (cytosol) fraction. A series of double immunoprecipitations with 12CA5 and polyclonal antibodies against Kv1.5 and Kv1.4 revealed that Kv1.1N206Tag forms heteromultimeric complexes with the native Kv1.4 and Kv1.5 polypeptides. The steady-state levels of Kv1.5 were not affected by the overexpression of Kv1.1N206Tag. Immunofluorescence colocalization and confocal microscopy analyses revealed that Kv1.1N206TagFlag did not reach the plasma membrane, and its distribution pattern was characteristic to that of a resident endoplasmic reticulum polypeptide. Our observations establish that the negative effect of Kv1.1N206Tag is mediated by the formation of heteromultimeric complexes with the native channels and by the retention of these complexes in the endoplasmic reticulum.

A protein fragment of streptococcal cell surface antigen I/II which prevents adhesion of Streptococcus mutans.

Attachment of Streptococcus mutans to the tooth surface involves a cell surface protein with an M(r) of 185,000, termed streptococcal antigen (SA) I/II. Four overlapping fragments of the gene encoding SA I/II were amplified by polymerase chain reaction, cloned, and expressed in Escherichia coli. The recombinant polypeptides were assayed for adhesion-binding activity to salivary receptors and for recognition by a panel of monoclonal antibodies (MAbs) raised against SA I/II. Two of the MAbs which are known to prevent colonization of S. mutans in vivo bound the recombinant polypeptide comprising residues 816 to 1161. In vitro adhesion of S. mutans to saliva-coated hydroxyapatite beads was also inhibited specifically by a polypeptide (residues 816 to 1213) encompassing the same region. The evidence from the MAbs preventing colonization of S. mutans and the adherence inhibition assay suggests that an adhesion-binding activity resides within the portion of SA I/II comprising residues 816 to 1213, which is highly conserved among oral streptococcal species.