Kv4 channel complexes mediate the neuronal somatodendritic A-type K(+) current (I(SA)), which plays pivotal roles in dendritic signal integration. These complexes are composed of pore-forming voltage-gated alpha-subunits (Shal/Kv4) and at least two classes of auxiliary beta-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 beta-subunits. Namely, we have concluded that: (1) the Kv4 channel complex employs novel alternative mechanisms of closed-state inactivation; (2) the intracellular Zn(2+) 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 I(SA) function and regulation.

The cyclic lipodepsipeptide, syringomycin E, when incorporated into planar lipid bilayer membranes, forms two types of channels (small and large) that are different in conductance by a factor of sixfold. To discriminate between a cluster organization-type channel structure and other possible different structures for the two channel types, their ionic selectivity and pore size were determined. Pore size was assessed using water-soluble polymers. Ion selectivity was found to be essentially the same for both the small and large channels. Their reversal (zero current) potentials with the sign corresponding to anionic selectivity did not differ by more than 3 mV at a twofold electrolyte gradient across the bilayer. Reduction in the single-channel conductance induced by poly(ethylene glycol)s of different molecular weights demonstrated that the aqueous pore sizes of the small and large channels did not differ by more than 2% and were close to 1 nm. Based on their virtually identical selectivity and size, we conclude that large syringomycin E channels are clusters of small ones exhibiting synchronous opening and closing.

The neuronal subthreshold-operating A-type K+ current regulates electrical excitability, spike timing and synaptic integration and plasticity. The Kv4 channels underlying this current have been implicated in epilepsy, regulation of dopamine release, and pain plasticity. However, the unitary conductance (γ) of neuronal somatodendritic A-type K+ channels composed of Kv4 pore-forming subunits is larger (∼7.5 pS) than that of Kv4 channels expressed singly in heterologous cells (∼4 pS). Here, we examined the putative novel contribution of the dipeptidyl-peptidase-like-protein-6 DPP6-S to the γ of native (cerebellar granule neuron, CGN) and reconstituted Kv4.2 channels. Co-expression of Kv4.2 proteins with DPP6-S was sufficient to match the γ of native CGN channels; and CGN Kv4 channels from dpp6 knock-out mice yielded a γ indistinguishable from that of Kv4.2 channels expressed singly. Moreover, suggesting electrostatic interactions, charge neutralization mutations of two N-terminal acidic residues in DPP6-S eliminated the increase in γ. Therefore, DPP6-S, as a membrane protein extrinsic to the pore domain, is necessary and sufficient to explain a fundamental difference between native and recombinant Kv4 channels. These observations may help to understand the molecular basis of neurological disorders correlated with recently identified human mutations in the dpp6 gene.

We evaluated the effect of agents modifying the membrane dipole potential: phloretin, 6-ketocholestanol and RH 421 on the properties of single channels formed by lipodepsipeptide syringomycin E (SRE) in planar lipid bilayers. SRE forms two conductive states in lipid bilayers: "small" and "large." Large SRE channels are clusters of several small ones, demonstrating synchronous openings and closures. The increase in the membrane dipole potential led to (i) an increase in SRE channel conductance, (ii) an increase in the channel's lifetime, and (iii) a decrease in a number of synchronously operating small channels in the clusters. Overall, the results support the model of the small SRE channel synchronization in the cluster as voltage-dependent orientation of the lipid dipoles associated with the channel pores.

The Smith-Lemli-Opitz syndrome (SLOS) is an inherited disorder of cholesterol synthesis caused by mutations in DHCR7 which encodes the final enzyme in the cholesterol synthesis pathway. The immediate precursor to cholesterol synthesis, 7-dehydrocholesterol (7-DHC) accumulates in the plasma and cells of SLOS patients which has led to the idea that the accumulation of abnormal sterols and/or reduction in cholesterol underlies the phenotypic abnormalities of SLOS. We tested the hypothesis that 7-DHC accumulates in membrane caveolae where it disturbs caveolar bilayer structure-function. Membrane caveolae from skin fibroblasts obtained from SLOS patients were isolated and found to accumulate 7-DHC. In caveolar-like model membranes containing 7-DHC, subtle, but complex alterations in intermolecular packing, lipid order and membrane width were observed. In addition, the BKCa K+ channel, which co-migrates with caveolin-1 in a membrane fraction enriched with cholesterol, was impaired in SLOS cells as reflected by reduced single channel conductance and a 50 mV rightward shift in the channel activation voltage. In addition, a marked decrease in BKCa protein but not mRNA expression levels were seen suggesting post-translational alterations. Accompanying these changes was a reduction in caveolin-1 protein and mRNA levels, but membrane caveolar structure was not altered. These results are consistent with the hypothesis that 7-DHC accumulation in the caveolar membrane results in defective caveolar signaling. However, additional cellular alterations beyond mere changes associated with abnormal sterols in the membrane likely contribute to the pathogenesis of SLOS.

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The neuronal subthreshold-operating A-type K ϩ current regulates electrical excitability, spike timing, and synaptic integration and plasticity. The Kv4 channels underlying this current have been implicated in epilepsy, regulation of dopamine release, and pain plasticity. However, the unitary conductance (␥) of neuronal somatodendritic A-type K ϩ channels composed of Kv4 pore-forming subunits is larger (ϳ7.5 pS) than that of Kv4 channels expressed singly in heterologous cells (ϳ4 pS). Here, we examined the putative novel contribution of the dipeptidyl-peptidase-like protein-6 DPP6-S to the ␥ of native [cerebellar granule neuron (CGN)] and reconstituted Kv4.2 channels. Coexpression of Kv4.2 proteins with DPP6-S was sufficient to match the ␥ of native CGN channels; and CGN Kv4 channels from dpp6 knockout mice yielded a ␥ indistinguishable from that of Kv4.2 channels expressed singly. Moreover, suggesting electrostatic interactions, charge neutralization mutations of two N-terminal acidic residues in DPP6-S eliminated the increase in ␥. Therefore, DPP6-S, as a membrane protein extrinsic to the pore domain, is necessary and sufficient to explain a fundamental difference between native and recombinant Kv4 channels. These observations may help to understand the molecular basis of neurological disorders correlated with recently identified human mutations in the dpp6 gene.

Sphingolipids with long chain bases hydroxylated at the C4 position are a requisite for the yeast, Saccharomyces cerevisia, to be sensitive to the ion channel forming antifungal agent, syringomycin E (SRE). A mutant S. cerevisiae strain, Deltasyr2, having sphingolipids with a sphingoid base devoid of C4-hydroxylation, is resistant to SRE. To explore the mechanism of this resistance, we investigated the channel forming activity of SRE in lipid bilayers of varying composition. We found that the addition of sphingolipid-rich fraction from Deltasyr2 to the membrane-forming solution (DOPS/DOPE/ergosterol) resulted in lipid bilayers with lower sensitivity to SRE compared with those containing sphingolipid fraction from wild-type S. cerevisiae. Other conditions being equal, the rate of increase of bilayer conductance was about 40 times slower, and the number of SRE channels was about 40 times less, with membranes containing Deltasyr2 versus wild-type sphingolipids. Deltasyr2 sphingolipids altered neither SRE single channel conductance nor the gating charge but the ability of SRE channels to open synchronously was diminished. The results suggest that the resistance of the Deltasyr2 mutant to SRE may be partly due to the ability of sphingolipids without the C4 hydroxyl group to decrease the channel forming activity of SRE.