Attenuation of the Slow Component of Delayed Rectification, Action Potential Prolongation, and Triggered Activity in Mice Expressing a Dominant-Negative Kv2 α Subunit

Abstract

An in vivo experimental strategy, involving cardiac-specific expression of a mutant Kv 2.1 subunit that functions as a dominant negative, was exploited in studies focused on exploring the role of members of the Kv2 subfamily of pore-forming (alpha) subunits in the generation of functional voltage-gated K(+) channels in the mammalian heart. A mutant Kv2.1 alpha subunit (Kv2.1N216) was designed to produce a truncated protein containing the intracellular N terminus, the S1 membrane-spanning domain, and a portion of the S1/S2 loop. The truncated Kv2.1N216 was epitope tagged at the C terminus with the 8-amino acid FLAG peptide to generate Kv2. 1N216FLAG. No ionic currents are detected on expression of Kv2. 1N216FLAG in HEK-293 cells, although coexpression of this construct with wild-type Kv2.1 markedly reduced the amplitudes of Kv2. 1-induced currents. Using the alpha-myosin heavy chain promoter to direct cardiac specific expression of the transgene, 2 lines of Kv2. 1N216FLAG-expressing transgenic mice were generated. Electrophysiological recordings from ventricular myocytes isolated from these animals revealed that I(K, slow) is selectively reduced. The attenuation of I(K, slow) is accompanied by marked action potential prolongation, and, occasionally, spontaneous triggered activity (apparently induced by early afterdepolarizations) is observed. The time constant of inactivation of I(K, slow) in Kv2. 1N216FLAG-expressing cells (mean+/-SEM=830+/-103 ms; n=17) is accelerated compared with the time constant of I(K, slow) inactivation (mean+/-SEM=1147+/-57 ms; n=25) in nontransgenic cells. In addition, unlike I(K, slow) in wild-type cells, the component of I(K, slow) remaining in the Kv2.1N216FLAG-expressing cells is insensitive to 25 mmol/L tetraethylammonium. Taken together, these observations suggest that there are 2 distinct components of I(K, slow) in mouse ventricular myocytes and that Kv2 alpha subunits underlie the more slowly inactivating, tetraethylammonium-sensitive component of I(K, slow). In vivo telemetric recordings also reveal marked QT prolongation, consistent with a defect in ventricular repolarization, in Kv2.1N216FLAG-expressing transgenic mice.

Elimination of the Fast Transient in Superior Cervical Ganglion Neurons with Expression of KV4.2W362F: Molecular Dissection ofIA

“…5), it seems reasonable to suggest that there are two distinct types of I Af channels in SCG neurons that reflect the functional expression of distinct gene products. Interestingly, the identification of two molecularly distinct components of a "single" electrophysiologically defined current has been reported previously for I Kr in Xenopus spinal neurons (Ribera, 1996) and for I K , slow in mouse ventricular myocytes (Xu et al, 1999b). Recent studies have also revealed two components of recovery of I Af from steady-state inactivation with time constants of ϳ100 and ϳ1000 msec (n ϭ 2), observations consistent with the suggestion that there are two components I Af .…”

Functional antagonism of voltage-gated K+ channel α-subunits in the developing brain ventricular system

“…6D) at 40 mV, where the open probability of both K v 2.1/ Kcng4b and K v 2.1/Kcng4b* channels is 1, excluding the possibility that the differences in current density reflect the difference in open probability. This suggests that Kcng4b* does interact with K v 2.1 and exerts a dominant-negative effect, as has been demonstrated for the dominant-negative mutants of K v 1.5 and K v 2.1 (Babila et al, 1994;Xu et al, 1999). Taken together, the developmental and electrophysiological analysis of the kcng4b mutant demonstrated that the mutant phenotype resulting from the LOF of Kcng4b has been further compounded by the dominant-negative effect of Kcng4b*.…”

Left Ventricular Hypertrophy

“…The expression of transcripts encoding I K1 channel ␣ subunits Kir2.1 (KCNJ2) and Kir2.2 (KCNJ12), 23 and of the K2P channel subunit TASK1 (KCNK3), which has been suggested to underlie I ss in rat cardiomyocytes, 26 as well as TASK2 (KCNK5), was unaffected in TAC LV ( Figure 5C). In contrast, expression of the transcripts encoding I K,slow1 and I K,slow2 channels Kv1.5 (KCNA5) and Kv2.1 (KCNB1), 14,22 as well as of the K2P channel subunit TREK1 (KCNK2), was increased in TAC LV ( Figure 5C). The expression levels of several other Kv ␣ subunits Kv1.4 (KCNA4), Kv4.1 (KCND1), and KvLQT1 (KCNQ1), as well as a number of K ϩ channel regulatory proteins including Kv␤1 (KCNAB1), Kv␤2 (KCNAB2), minK (KCNE1), KChAP (Pias3), PSD-95 (post-synaptic density 95 protein), and filamin C, were also increased in TAC LV ( Figure 5C).…”

“…14,19,[22][23][24] Because accumulating evidence suggests that cardiac ion channels function as components of macromolecular complexes, 25 TaqMan low-density arrays 16 were exploited to allow quantitative determinations of multiple transcripts simultaneously. The amount of total RNA isolated from TAC LV was significantly (PϽ0.01) higher (1.7-fold on average) than from sham LV ( Figure 5A).…”