Differential modulation of Kv4.2 and Kv4.3 channels by calmodulin-dependent protein kinase II in rat cardiac myocytes

Abstract

In this work we have combined biochemical and electrophysiological approaches to explore the modulation of rat ventricular transient outward K(+) current (I(to)) by calmodulin kinase II (CaMKII). Intracellular application of CaMKII inhibitors KN93, calmidazolium, and autocamtide-2-related inhibitory peptide II (ARIP-II) accelerated the inactivation of I(to), even at low [Ca(2+)]. In the same conditions, CaMKII coimmunoprecipitated with Kv4.3 channels, suggesting that phosphorylation of Kv4.3 channels modulate inactivation of I(to). Because channels underlying I(to) are heteromultimers of Kv4.2 and Kv4.3, we have explored the effect of CaMKII on human embryonic kidney (HEK) cells transfected with either of those Kvalpha-subunits. Whereas Kv4.3 inactivated faster upon inhibition of CaMKII, Kv4.2 inactivation was insensitive to CaMKII inhibitors. However, Kv4.2 inactivation became slower when high Ca(2+) was used in the pipette or when intracellular [Ca(2+)] ([Ca(2+)](i)) was transiently increased. This effect was inhibited by KN93, and Western blot analysis demonstrated Ca(2+)-dependent phosphorylation of Kv4.2 channels. On the contrary, CaMKII coimmunoprecipitated with Kv4.3 channels without a previous Ca(2+) increase, and the association was inhibited by KN93. These results suggest that both channels underlying I(to) are substrates of CaMKII, although with different sensitivities; Kv4.2 remain unphosphorylated unless [Ca(2+)](i) increases, whereas Kv4.3 are phosphorylated at rest. In addition to the functional impact that phosphorylation of Kv4 channels could cause on the shape of action potential, association of CaMKII with Kv4.3 provides a new role of Kv4.3 subunits as molecular scaffolds for concentrating CaMKII in the membrane, allowing Ca(2+)-dependent modulation by this enzyme of the associated Kv4.2 channels.

Kv channel-interacting proteins as neuronal and non-neuronal calcium sensors

“…Notably, in cardiomyocytes KChIP2 may form a complex with Cav1.2 [68,69], maybe in combination with Kv4 channels. Also, CaMKII, which seems to play a critical role in the Ca 2+ -dependent regulation of Kv4/KChIP channels [22], may participate in this cardiac signaling complex [94–96]. Finally, it has to be examined whether other sources of Ca 2+ , including transmitter-gated channels like N-methyl-D-aspartic acid (NMDA) receptors, or ER Ca 2+ channels like the ryanodine and the IP3 receptor, are able to acutely modulate the gating of Kv4/KChIP channel complexes.…”

W-7 modulates Kv4.3: pore block and Ca2+-calmodulin inhibition

“…KN-93 competitively inhibits Ca 2ϩ -CaM binding to Ca 2ϩ /CaMKII (5, 7, 31a). Although prior studies examined the effects of W-7 and KN-93 on the native Ca 2ϩ and K ϩ currents in cardiac myocytes, the effects of these two agents on the gating and permeation properties of K v 4 channels have not been analyzed in detail (5,19,38,42,60,69).…”

“…In addition, the effects of KN-93 on the A-type current in the central nervous system and smooth muscle of the gastrointestinal tract have been studied (1,2,11,35,39,40). Ca 2ϩ /CaMKII activation and inhibition have been shown to modulate A-type current in colonic and gastric myocytes and in cardiac myocytes (1,2,19,39,40). Sergeant et al (63) demonstrated that KN-93 and a constitutively active Ca 2ϩ / CaMKII modulated inactivation, recovery from inactivation, and the steady-state inactivation relation of the heterologously expressed K v 4.3 channel.…”

“…Although the effects of KN-93 on the K v 4.3 channel were reported to modify kinetics of inactivation and recovery (19,63), these studies did not address whether some of the effects of KN-93 on peak current were also due to open-channel block, i.e., were voltage dependent. KN-93 at 35, 50, and 100 M reduced peak current at ϩ50 mV by 4.9 Ϯ 3.5%, 14.4 Ϯ 5.9%, and 30.3 Ϯ 3.5%, respectively (Fig.…”

“…Agents that directly decrease the activity of Ca 2ϩ /CaMKII have been shown to accelerate inactivation kinetics of heterologously expressed K v 4.3 channels (19,63). W-7 inhibits Ca 2ϩ -CaM effects, which in turn decrease the activity of Ca 2ϩ /CaMKII as well as other kinases and phosphatases.…”