The Electrically Silent Kv6.4 Subunit Confers Hyperpolarized Gating Charge Movement in Kv2.1/Kv6.4 Heterotetrameric Channels

The voltage-gated K+ (Kv) channel subunit Kv6.4 does not form functional homotetrameric channels but co-assembles with Kv2.1 to form functional Kv2.1/Kv6.4 heterotetrameric channels. Compared to Kv2.1 homotetramers, Kv6.4 exerts a ∼40 mV hyperpolarizing shift in the voltage-dependence of Kv2.1/Kv6.4 channel inactivation, without a significant effect on activation gating. However, the underlying mechanism of this Kv6.4-induced modulation of Kv2.1 channel inactivation, and whether the Kv6.4 subunit participates in the voltage-dependent gating of heterotetrameric channels is not well understood. Here we report distinct gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels, compared to Kv2.1 homotetramers, as revealed by gating current recordings from mammalian cells expressing these channels. The gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels displayed an extra component around the physiological K+ equilibrium potential, characterized by a second sigmoidal relationship of the voltage-dependence of gating charge movement. This distinct gating charge displacement reflects movement of the Kv6.4 voltage-sensing domain and has a voltage-dependency that matches the hyperpolarizing shift in Kv2.1/Kv6.4 channel inactivation. These results provide a mechanistic basis for the modulation of Kv2.1 channel inactivation gating kinetics by silent Kv6.4 subunits

The C-type natriuretic peptide induces thermal hyperalgesia through a noncanonical Gβγ-dependent modulation of TRPV1 channel

Natriuretic peptides (NPs) control natriuresis and normalize changes in blood pressure. Recent studies suggest that NPs are also involved in the regulation of pain sensitivity, although the underlying mechanisms remain largely unknown. Many biological effects of NPs are mediated by guanylate cyclase (GC)-coupled NP receptors, NPR-A and NPR-B, whereas the third NP receptor, NPR-C, lacks the GC kinase domain and acts as the NP clearance receptor. In addition, NPR-C can couple to specific Gα(i)-βγ-mediated intracellular signaling cascades in numerous cell types. We found that NPR-C is co-expressed in TRPV1-expressing mouse DRG neurons. NPR-C can be co-immunoprecipitated with Gα(i), and CNP treatment induced translocation of PKCε to the plasma membrane of these neurons, which was inhibited by pertussis toxin pre-treatment. Application of CNP potentiated capsaicin- and proton-activated TRPV1 currents in cultured mouse DRG neurons, and increased neuronal firing frequency, an effect that was absent in DRG neurons from TRPV1(−/−) mice. CNP-induced sensitization of TRPV1 activity was attenuated by pre-treatment of DRG neurons with the specific inhibitors of Gβγ, PLCβ or PKC, but not of PKA, and was abolished by mutations at two PKC phosphorylation sites in TRPV1. Further, CNP injection into mouse hind paw led to the development of thermal hyperalgesia that was attenuated by administration of specific inhibitors of Gβγ or TRPV1, and was also absent in TRPV1(−/−) mice. Thus, our work identifies the Gβγ-PLCβ-PKC-dependent potentiation of TRPV1 as a novel signaling cascade recruited by CNP in mouse DRG neurons that can lead to enhanced nociceptor excitability and thermal hypersensitivity

Competition between α-actinin and Ca2+-Calmodulin Controls Surface Retention of the L-type Ca2+ Channel CaV1.2

Regulation of neuronal excitability and cardiac excitation-contraction coupling requires proper localization of L-type Ca2+ channels. We show that the actin-binding protein α-actinin binds to the C-terminal surface targeting motif of α11.2, the central pore-forming CaV1.2 subunit, to foster its surface expression. Disruption of α-actinin function by dominant negative or shRNA constructs reduces CaV1.2 surface localization in HEK293 and neuronal cultures, and dendritic spine localization in neurons. We demonstrate that calmodulin displaces α-actinin from their shared binding site on α11.2 upon Ca2+ influx through L-type channels but not through NMDAR, thereby triggering loss of CaV1.2 from spines. Coexpression of a Ca2+-binding deficient calmodulin mutant does not affect basal CaV1.2 surface expression, but inhibits its internalization upon Ca2+ influx. We conclude that α-actinin stabilizes CaV1.2 at the plasma membrane, and that its displacement by Ca2+-calmodulin induces Ca2+-induced endocytosis of CaV1.2, thus providing an important negative feedback mechanism for Ca2+ influx

Parathyroid Hormone-Related Peptide Elicits Peripheral TRPV1-dependent Mechanical Hypersensitivity

Bone metastasis in breast, prostate and lung cancers often leads to chronic pain, which is poorly managed by existing analgesics. The neurobiological mechanisms that underlie chronic pain associated with bone-metastasized cancers are not well understood, but sensitization of peripheral nociceptors by tumor microenvironment factors has been demonstrated to be important. Parathyroid hormone-related peptide (PTHrP) is highly expressed in bone-metastasized breast and prostate cancers, and is critical to growth and proliferation of these tumors in the bone tumor microenvironment. Previous studies have suggested that PTHrP could sensitize nociceptive sensory neurons, resulting in peripheral pain hypersensitivity. In this study, we found that PTHrP induces both heat and mechanical hypersensitivity, that are dependent on the pain-transducing transient receptor potential channel family vanilloid, member-1 (TRPV1), but not the mechano-transducing TRPV4 and TRPA1 ion channels. Functional ratiometric Ca2+ imaging and voltage-clamp electrophysiological analysis of cultured mouse DRG neurons show significant potentiation of TRPV1, but not TRPA1 or TRPV4 channel activation by PTHrP. Interestingly, PTHrP exposure led to the slow and sustained activation of TRPV1, in the absence of any exogenous channel agonist, and is dependent on the expression of the type-1 parathyroid hormone receptor (PTH1), as well as on downstream phosphorylation of the channel by protein kinase C (PKC). Accordingly, local administration of specific small-molecule antagonists of TRPV1 to mouse hindpaws after the development of PTHrP-induced mechanical hypersensitivity led to its significant attenuation. Collectively, our findings suggest that PTHrP/PTH1-mediated flow activation of TRPV1 channel contributes at least in part to the development and maintenance of peripheral mechanical pain hypersensitivity, and could therefore constitute a mechanism for nociceptor sensitization in the context of metastatic bone cancer pain

Chemokine co-receptor CCR5/CXCR4-dependent modulation of Kv2.1 channel confers acute neuroprotection to HIV-1 glycoprotein gp120 exposure.

Infection with human immunodeficiency virus-1 (HIV-1) within the brain has long been known to be associated with neurodegeneration and neurocognitive disorder (referred as HAND), a condition characterized in its early stages by declining cognitive function and behavioral disturbances. Mechanistically, the HIV-1 coat glycoprotein 120 (gp120) has been suggested to be a critical factor inducing apoptotic cell death in neurons via the activation of p38 mitogen-activated protein kinase (MAPK), upon chronic exposure to the virus. Here we show that acute exposure of neurons to HIV-1 gp120 elicits a homeostatic response, which provides protection against non-apoptotic cell death, involving the major somatodendritic voltage-gated K⁺ (Kv) channel Kv2.1 as the key mediator. The Kv2.1 channel has recently been shown to provide homeostatic control of neuronal excitability under conditions of seizures, ischemia and neuromodulation/neuroinflammation. Following acute exposure to gp120, cultured rat hippocampal neurons show rapid dephosphorylation of the Kv2.1 protein, which ultimately leads to changes in specific sub-cellular localization and voltage-dependent channel activation properties of Kv2.1. Such modifications in Kv2.1 are dependent on the activation of the chemokine co-receptors CCR5 and CXCR4, and subsequent activation of the protein phosphatase calcineurin. This leads to the overall suppression of neuronal excitability and provides neurons with a homeostatic protective mechanism. Specific blockade of calcineurin and Kv2.1 channel activity led to significant enhancement of non-apoptotic neuronal death upon acute gp120 treatment. These observations shed new light on the intrinsic homeostatic mechanisms of neuronal resilience during the acute stages of neuro-HIV infections