In the brain and heart, rapidly inactivating (A-type) voltage-gated potassium (Kv) currents operate at subthreshold membrane potentials to control the excitability of neurons and cardiac myocytes. Although pore-forming alpha-subunits of the Kv4, or Shal-related, channel family form A-type currents in heterologous cells, these differ significantly from native A-type currents. Here we describe three Kv channel-interacting proteins (KChIPs) that bind to the cytoplasmic amino termini of Kv4 alpha-subunits. We find that expression of KChIP and Kv4 together reconstitutes several features of native A-type currents by modulating the density, inactivation kinetics and rate of recovery from inactivation of Kv4 channels in heterologous cells. All three KChIPs co-localize and co-immunoprecipitate with brain Kv4 alpha-subunits, and are thus integral components of native Kv4 channel complexes. The KChIPs have four EF-hand-like domains and bind calcium ions. As the activity and density of neuronal A-type currents tightly control responses to excitatory synaptic inputs, these KChIPs may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium.

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Calcium‐binding proteins dubbed KChIPs favour surface expression and modulate inactivation gating of neuronal and cardiac A‐type Kv4 channels. To investigate their mechanism of action, Kv4.1 or Kv4.3 were expressed in Xenopus laevis oocytes, either alone or together with KChIP1, and the K+ currents were recorded using the whole‐oocyte voltage‐clamp and patch‐clamp methods. KChIP1 similarly remodels gating of both channels. At positive voltages, KChIP1 slows the early phase of the development of macroscopic inactivation. By contrast, the late phase is accelerated, which allows complete inactivation in < 500 ms. Thus, superimposed traces from control and KChIP1‐remodelled currents crossover. KChIP1 also accelerates closed‐state inactivation and recovery from inactivation (3‐ to 5‐fold change). The latter effect is dominating and, consequently, the prepulse inactivation curves exhibit depolarizing shifts (ΔV= 4–12 mV). More favourable closed‐state inactivation may also contribute to the overall faster inactivation at positive voltages because Kv4 channels significantly inactivate from the preopen closed state. KChIP1 favours this pathway further by accelerating channel closing. The peak G‐V curves are modestly leftward shifted in the presence of KChIP1, but the apparent ‘threshold’ voltage of current activation remains unaltered. Single Kv4.1 channels exhibited multiple conductance levels that ranged between 1.8 and 5.6 pS in the absence of KChIP1 and between 1.9 and 5.3 pS in its presence. Thus, changes in unitary conductance do not contribute to current upregulation by KChIP1. An allosteric kinetic model explains the kinetic changes by assuming that KChIP1 mainly impairs open‐state inactivation, favours channel closing and lowers the energy barrier of closed‐state inactivation.

Protein tyrosine kinases (PTKs) coordinate a broad spectrum of cellular responses to extracellular stimuli and cell–cell interactions during development, tissue homeostasis, and responses to environmental challenges. Thus, an understanding of the regulatory mechanisms that ensure physiological PTK function and potential aberrations of these regulatory processes during diseases such as cancer are of broad interest in biology and medicine. Aside from the expected role of phospho-tyrosine phosphatases, recent studies have revealed a critical role of covalent modification of activated PTKs with ubiquitin as a critical mechanism of their negative regulation. Members of the Cbl protein family (Cbl, Cbl-b and Cbl-c in mammals) have emerged as dominant “activated PTK-selective” ubiquitin ligases. Structural, biochemical and cell biological studies have established that Cbl protein-dependent ubiquitination targets activated PTKs for degradation either by facilitating their endocytic sorting into lysosomes or by promoting their proteasomal degradation. This mechanism also targets PTK signaling intermediates that become associated with Cbl proteins in a PTK activation-dependent manner. Cellular and animal studies have established that the relatively broadly expressed mammalian Cbl family members Cbl and Cbl-b play key physiological roles, including their critical functions to prevent the transition of normal immune responses into autoimmune disease and as tumor suppressors; the latter function has received validation from human studies linking mutations in Cbl to human leukemia. These newer insights together with embryonic lethality seen in mice with a combined deletion of Cbl and Cbl-b genes suggest an unappreciated role of the Cbl family proteins, and by implication the ubiquitin-dependent control of activated PTKs, in stem/progenitor cell maintenance. Future studies of existing and emerging animal models and their various cell lineages should help test the broader implications of the evolutionarily-conserved Cbl family protein-mediated, ubiquitin-dependent, negative regulation of activated PTKs in physiology and disease.

The Kv4 A-type potassium currents contribute to controlling the frequency of slow repetitive firing and back-propagation of action potentials in neurons and shape the action potential in heart. Kv4 currents exhibit rapid activation and inactivation and are specifically modulated by K-channel interacting proteins (KChIPs). Here we report the discovery and functional characterization of a modular K-channel inactivation suppressor (KIS) domain located in the first 34 aa of an additional KChIP (KChIP4a T he Kv4 subfamily of voltage-gated potassium channels underlie somatodendritic A-currents in several types of neurons (1-3) and I to in cardiac myocytes (4-7). Operating at subthreshold membrane potentials, they contribute to controlling the frequency of slow repetitive firing in these excitable cells. The dendritic A-type K ϩ current in hippocampal neurons helps to integrate the back-propagating action potentials and excitatory postsynaptic potentials or inhibitory postsynaptic potentials, providing a rapid electric signal to initiate associative events such as long-term potentiation (LTP) and long-term depression (LDP) (8-12). In heart, I to impacts on the early phase of repolarization of the action potential (13,14).We recently identified K-channel interacting protein 1-3 (KChIP1-3) that specifically modulate Kv4 currents (15). KChIP1-3 increase total Kv4 current, moderately slow channel inactivation, and considerably accelerate recovery from inactivation (15). They are EF-hand Ca 2ϩ -binding proteins that belong to the recoverin͞neuronal calcium sensor-1 (NCS-1) family. KChIP1 and KChIP 3 are predominantly expressed in neuronal tissues, whereas KChIP2 is predominantly expressed in heart and brain (15).Here we report an unexpected, distinct modulation of Kv4 currents by a K-channel inactivation suppressor (KIS) domain present in an additional KChIP, KChIP4a. We show that by eliminating fast inactivation in conjunction with changes in other kinetic parameters, the KIS domain effectively converts the fast inactivating A-type current to a slowly inactivating delayed rectifier type of currents. Also, we present evidence that KChIPs with and without the KIS domain modulate Kv4 currents in a combinatorial manner. The KIS domain acts oppositely to the Kv␤1 ball domain (16-19) and the ball-like domains of maxi-K ␤2, ␤3 subunits (20-22). These observations indicate that auxiliary subunits provide diverse mechanisms to control activity of potassium channels. Materials and MethodsElectrophysiology. Unitary potassium currents were recorded from cell-attached patches in the presence of 2 mM KCl in the recording pipette as described (23, 24). Macroscopic potassium currents were recorded by applying the two-electrode voltage clamp method in Xenopus oocytes and the tight-seal whole-cell method in Chinese hamster ovary cells and cerebellar granule neurons essentially as described (25), except noted as follows. To examine the kinetics of the macroscopic rising phase, the currents were evoked from a holding potential of Ϫ100 mV by 3...