Manuel Covarrubias

Molecular physiology and modulation of somatodendritic A-type potassium channels

The somatodendritic subthreshold A-type K+ current (ISA) in nerve cells is a critical component of the ensemble of voltage-gated ionic currents that determine somatodendritic signal integration. The underlying K+ channel belongs to the Shal subfamily of voltage-gated K+ channels. Most Shal channels across the animal kingdom share a high degree of structural conservation, operate in the subthreshold range of membrane potentials, and exhibit relatively fast inactivation and recovery from inactivation. Mammalian Shal K+ channels (Kv4) undergo preferential closed-state inactivation with features that are generally inconsistent with the classical mechanisms of inactivation typical of Shaker K+ channels. Here, we review (1) the physiological and genetic properties of ISA, 2 the molecular mechanisms of Kv4 inactivation and its remodeling by a family of soluble calcium-binding proteins (KChIPs) and a membrane-bound dipeptidase-like protein (DPPX), and (3) the modulation of Kv4 channels by protein phosphorylation.

Elimination of fast inactivation in Kv4 A-type potassium channels by an auxiliary subunit domain.

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). Coexpression of KChIP4a with Kv4 α-subunits abolishes fast inactivation of the Kv4 currents in various cell types, including cerebellar granule neurons. Kinetic analysis shows that the KIS domain delays Kv4.3 opening, but once the channel is open, it disrupts rapid inactivation and slows Kv4.3 closing. Accordingly, KChIP4a increases the open probability of single Kv4.3 channels. The net effects of KChIP4a and KChIP1–3 on Kv4 gating are quite different. When both KChIP4a and KChIP1 are present, the Kv4.3 current shows mixed inactivation profiles dependent on KChIP4a/KChIP1 ratios. The KIS domain effectively converts the A-type Kv4 current to a slowly inactivating delayed rectifier-type potassium current. This conversion is opposite to that mediated by the Kv1-specific “ball” domain of the Kvβ1 subunit. Together, these results demonstrate that specific auxiliary subunits with distinct functions actively modulate gating of potassium channels that govern membrane excitability.

Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate

The effect of protein kinase C (PKC) on rapid N-type inactivation of K+ channels has not been reported previously. We found that PKC specifically eliminates rapid inactivation of a cloned human A-type K+ channel (hKv3.4), converting this channel from a rapidly inactivating A type to a noninactivating delayed rectifier type. Biochemical analysis showed that the N-terminal domain of hKv3.4 is phosphorylated in vitro by PKC, and mutagenesis experiments revealed that two serines within the inactivation gate at the N-terminus are sites of direct PKC action. Moreover, mutating one of these serines to aspartic acid mimics the action of PKC. Serine phosphorylation may thus prevent rapid inactivation by shielding basic residues known to be critical to the function of the inactivation gate. The regulatory mechanism reported here may have substantial effects on signal coding in the nervous system.

K+ channel inactivation mediated by the concerted action of the cytoplasmic N- and C-terminal domains.

We have examined the molecular mechanism of rapid inactivation gating in a mouse Shal K+ channel (mKv4.1). The results showed that inactivation of these channels follows a complex time course that is well approximated by the sum of three exponential terms. Truncation of an amphipathic region at the N-terminus (residues 2-71) abolished the rapid phase of inactivation (r = 16 ms) and altered voltage-dependent gating. Surprisingly, these effects could be mimicked by deletions affecting the hydrophilic C-terminus. The sum of two exponential terms was sufficient to describe the inactivation of deletion mutants. In fact, the time constants corresponded closely to those of the intermediate and slow phases of inactivation observed with wild-type channels. Further analysis revealed that several basic amino acids at the N-terminus do not influence inactivation, but a positively charged domain at the C-terminus (amino acids 420-550) is necessary to support rapid inactivation. Thus, the amphipathic N-terminus and the hydrophilic C-terminus of mKv4.1 are essential determinants of inactivation gating and may interact with each other to maintain the N-terminal inactivation gate near the inner mouth of the channel. Furthermore, this inactivation gate may not behave like a simple open-channel blocker because channel blockade by internal tetraethylammonium was not associated with slower current decay and an elevated external K+ concentration retarded recovery from inactivation.

Remodelling inactivation gating of Kv4 channels by KChIP1, a small-molecular-weight calcium-binding protein

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.

Mechanisms of closed-state inactivation in voltage-gated ion channels

Abstract  Inactivation of voltage‐gated ion channels is an intrinsic auto‐regulatory process necessary to govern the occurrence and shape of action potentials and establish firing patterns in excitable tissues. Inactivation may occur from the open state (open‐state inactivation, OSI) at strongly depolarized membrane potentials, or from pre‐open closed states (closed‐state inactivation, CSI) at hyperpolarized and modestly depolarized membrane potentials. Voltage‐gated Na+, K+, Ca2+ and non‐selective cationic channels utilize both OSI and CSI. Whereas there are detailed mechanistic descriptions of OSI, much less is known about the molecular basis of CSI. Here, we review evidence for CSI in voltage‐gated cationic channels (VGCCs) and recent findings that shed light on the molecular mechanisms of CSI in voltage‐gated K+ (Kv) channels. Particularly, complementary observations suggest that the S4 voltage sensor, the S4S5 linker and the main S6 activation gate are instrumental in the installment of CSI in Kv4 channels. According to this hypothesis, the voltage sensor may adopt a distinct conformation to drive CSI and, depending on the stability of the interactions between the voltage sensor and the pore domain, a closed‐inactivated state results from rearrangements in the selectivity filter or failure of the activation gate to open. Kv4 channel CSI may efficiently exploit the dynamics of the subthreshold membrane potential to regulate spiking properties in excitable tissues.

The Neuronal Kv4 Channel Complex

Kv4 channel complexes mediate the neuronal somatodendritic A-type K+ current (ISA), which plays pivotal roles in dendritic signal integration. These complexes are composed of pore-forming voltage-gated α-subunits (Shal/Kv4) and at least two classes of auxiliary β-subunits: KChIPs (K+-Channel-Interacting-Proteins) and DPLPs (Dipeptidyl-Peptidase-Like-Proteins). Here, we review our investigations of Kv4 gating mechanisms and functional remodeling by specific auxiliary β-subunits. Namely, we have concluded that: (1) the Kv4 channel complex employs novel alternative mechanisms of closed-state inactivation; (2) the intracellular Zn2+ site in the T1 domain undergoes a conformational change tightly coupled to voltage-dependent gating and is targeted by nitrosative modulation; and (3) discrete and specific interactions mediate the effects of KChIPs and DPLPs on activation, inactivation and permeation of Kv4 channels. These studies are shedding new light on the molecular bases of ISA function and regulation.

Ternary Kv4.2 channels recapitulate voltage‐dependent inactivation kinetics of A‐type K+ channels in cerebellar granule neurons

Kv4 channels mediate most of the somatodendritic subthreshold operating A‐type current (ISA) in neurons. This current plays essential roles in the regulation of spike timing, repetitive firing, dendritic integration and plasticity. Neuronal Kv4 channels are thought to be ternary complexes of Kv4 pore‐forming subunits and two types of accessory proteins, Kv channel interacting proteins (KChIPs) and the dipeptidyl‐peptidase‐like proteins (DPPLs) DPPX (DPP6) and DPP10. In heterologous cells, ternary Kv4 channels exhibit inactivation that slows down with increasing depolarization. Here, we compared the voltage dependence of the inactivation rate of channels expressed in heterologous mammalian cells by Kv4.2 proteins with that of channels containing Kv4.2 and KChIP1, Kv4.2 and DPPX‐S, or Kv4.2, KChIP1 and DPPX‐S, and found that the relation between inactivation rate and membrane potential is distinct for these four conditions. Moreover, recordings from native neurons showed that the inactivation kinetics of the ISA in cerebellar granule neurons has voltage dependence that is remarkably similar to that of ternary Kv4 channels containing KChIP1 and DPPX‐S proteins in heterologous cells. The fact that this complex and unique behaviour (among A‐type K+ currents) is observed in both the native current and the current expressed in heterologous cells by the ternary complex containing Kv4, DPPX and KChIP proteins supports the hypothesis that somatically recorded native Kv4 channels in neurons include both types of accessory protein. Furthermore, quantitative global kinetic modelling showed that preferential closed‐state inactivation and a weakly voltage‐dependent opening step can explain the slowing of the inactivation rate with increasing depolarization. Therefore, it is likely that preferential closed‐state inactivation is the physiological mechanism that regulates the activity of both ternary Kv4 channel complexes and native ISA‐mediating channels.

Kv4 Channels Exhibit Modulation of Closed-State Inactivation in Inside-Out Patches

The mechanisms of inactivation gating of the neuronal somatodendritic A-type K(+) current and the cardiac I(to) were investigated in Xenopus oocyte macropatches expressing Kv4.1 and Kv4.3 channels. Upon membrane patch excision (inside-out), Kv4.1 channels undergo time-dependent acceleration of macroscopic inactivation accompanied by a parallel partial current rundown. These changes are readily reversible by patch cramming, suggesting the influence of modulatory cytoplasmic factors. The consequences of these perturbations were investigated in detail to gain insights into the biophysical basis and mechanisms of inactivation gating. Accelerated inactivation at positive voltages (0 to +110 mV) is mainly the result of reducing the time constant of slow inactivation and the relative weight of the slow component of inactivation. Concomitantly, the time constants of closed-state inactivation at negative membrane potentials (-90 to -50 mV) are substantially decreased in inside-out patches. Deactivation is moderately accelerated, and recovery from inactivation and the peak G--V curve exhibit little or no change. In agreement with more favorable closed-state inactivation in inside-out patches, the steady-state inactivation curve exhibits a hyperpolarizing shift of approximately 10 mV. Closed-state inactivation was similarly enhanced in Kv4.3. An allosteric model that assumes significant closed-state inactivation at all relevant voltages can explain Kv4 inactivation gating and the modulatory changes.

Cortactin Is Required for N-cadherin Regulation of Kv1.5 Channel Function

The intercalated disc serves as an organizing center for various cell surface components at the termini of the cardiomyocyte, thus ensuring proper mechanoelectrical coupling throughout the myocardium. The cell adhesion molecule, N-cadherin, is an essential component of the intercalated disc. Cardiac-specific deletion of N-cadherin leads to abnormal electrical conduction and sudden arrhythmic death in mice. The mechanisms linking the loss of N-cadherin in the heart and spontaneous malignant ventricular arrhythmias are poorly understood. To investigate whether ion channel remodeling contributes to arrhythmogenesis in N-cadherin conditional knock-out (N-cad CKO) mice, cardiac myocyte excitability and voltage-gated potassium channel (Kv), as well as inwardly rectifying K+ channel remodeling, were investigated in N-cad CKO cardiomyocytes by whole cell patch clamp recordings. Action potential duration was prolonged in N-cad CKO ventricle myocytes compared with wild type. Relative to wild type, IK,slow density was significantly reduced consistent with decreased expression of Kv1.5 and Kv accessory protein, Kcne2, in the N-cad CKO myocytes. The decreased Kv1.5/Kcne2 expression correlated with disruption of the actin cytoskeleton and reduced cortactin at the sarcolemma. Biochemical experiments revealed that cortactin co-immunoprecipitates with Kv1.5. Finally, cortactin was required for N-cadherin-mediated enhancement of Kv1.5 channel activity in a heterologous expression system. Our results demonstrate a novel mechanistic link among the cell adhesion molecule, N-cadherin, the actin-binding scaffold protein, cortactin, and Kv channel remodeling in the heart. These data suggest that in addition to gap junction remodeling, aberrant Kv1.5 channel function contributes to the arrhythmogenic phenotype in N-cad CKO mice.