PrecisION hKv4.3/hKChIP2-HEK Recombinant Cell Line

PrecisION hKv4.3/hKChIP2-HEK Recombinant Cell Line

Upstate (Millipore)

2 mL

Recombinant HEK293 cell line expressing human Kv4.3 / human KChIP2 ion channel

Kv4 subunits are the main pore-forming proteins responsible for the fast transient outward currents observed in the CNS (rat brain), where they have been described as ‘A’ currents (IA) (Serodio and Rudy, 1998) and in mammalian heart, where they are known as Ca2+-independent fast outward currents (Ito,f.) (Nerbonne, 2000). Although in human heart the predominant subunit responsible for Ito,f is Kv4.3, in order to reconstitute all the properties of the native current co-expression with an auxiliary protein KChIP (Kv Channel Interacting Protein) is required. These small molecular weight Ca2+-binding proteins typically increase cell surface expression of the channel complex, accelerate the rate of decay of the current at depolarized potentials, and increase the rate of recovery from inactivation at hyperpolarized potentials, i.e. the kinetics then more closely resembles native Ito than if Kv4.3 subunits were expressed alone (Wang et al., 2002 and Deschenes et al., 2002). The predominant KChIP found in heart is KChIP2 (Rosati et al., 2001 and Deschenes et al., 2002) and can exist as various isoforms (Decher et al., 2004). The isoform co-expressed with Kv4.3 subunits in this cell line is KChiP2b (Decher et al., 2004) but will simply be referred to as KChIP2 in this document. In the heart, Ito,f is primarily responsible for the ‘notch’ during phase 1 of the cardiac action potential. Since it is an early repolarizing current it is of crucial importance in shaping the final cardiac action potential waveform (Nerbonne, 2000). In the human heart the density of Ito,f is highest in the epicardium and lowest in the endocardium; regional differences controlled by the level of KChiP2 expression (Rosati et al., 2001). These regional differences significantly contribute to the transmural voltage gradient across the myocardial wall, necessary for normal ventricular activity (Sanguinetti, 2002). Abolishing KChIP2 expression has been shown in mice to markedly affect this gradient with the consequence of increased susceptibility to arrhythmia (Kuo et al., 2001).

hKv4.3 has been co-expressed with KChIP2 in a HEK293 cell line to produce currents that more closely resemble native heart Ito,f. Currents have been characterized in terms of their biophysical properties and their sensitivity to phrixotoxin. Using whole-cell patch clamp techniques the threshold of current activation was around –40 mV and the peak current/voltage was linear where the mean peak amplitude at +40 mV was 13.8 ± 1.6 nA (n=6). The currents rapidly inactivated during the depolarizing pulse following a bi-exponential time course. These features are typical of Kv4 currents. Evoked currents were also sensitive to phrixotoxin-2 in the nanomolar range (IC50 around 30 nM) confirming the presence of Kv4 subunits. Co-expression of hKv4.3 with hKChIP2 had a marked effect on the rate of recovery from inactivation at negative potentials consistent with published reports. The time constant of recovery could be described by a single exponential with a time constant (τ) of 125 ± 14 ms (n=6). The rate of recovery was significantly slower when hKv4.3 was expressed alone, τ of 450 ± 25 ms (n = 6). Functional channel expression over time was monitored using IonWorks™ HT. Channel expression is robust over at least 41 passages. At passage 41, 99% of cells expressed hKv4.3/hKChIP2 currents >500 pA with a mean current amplitude of 2.74 ± 0.04 nA (n=178).

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Human

Mycoplasma Testing:
The cell line has been screened to confirm the absence of Mycoplasma species

HEK

Ion Channel Cell Lines

Pack contains 2 vials of mycoplasma-free cells, 1 ml per vial

Upstate

293 cell line

hKv4.3/hKChIP2

2 x 1 ml aliquots

Potassium

Ion Channels

PrecisION hKv4.3/hKChIP2-HEK Recombinant Cell Line

• Ito
• KCND3