Unmyelinated auditory type I spiral ganglion neurons in congenic Ly5.1 mice

With the exception of humans, the somata of type I spiral ganglion neurons (SGNs) of most mammalian species are heavily myelinated. In an earlier study, we used Ly5.1 congenic mice as transplant recipients to investigate the role of hematopoietic stem cells in the adult mouse inner ear. An unanticipated finding was that a large percentage of the SGNs in this strain were unmyelinated. Further characterization of the auditory phenotype of young adult Ly5.1 mice in the present study revealed several unusual characteristics, including 1) large aggregates of unmyelinated SGNs in the apical and middle turns, 2) symmetrical junction‐like contacts between the unmyelinated neurons, 3) abnormal expression patterns for CNPase and connexin 29 in the SGN clusters, 4) reduced SGN density in the basal cochlea without a corresponding loss of sensory hair cells, 5) significantly delayed auditory brainstem response (ABR) wave I latencies at low and middle frequencies compared with control mice with similar ABR threshold, and 6) elevated ABR thresholds and deceased wave I amplitudes at high frequencies. Taken together, these data suggest a defect in Schwann cells that leads to incomplete myelinization of SGNs during cochlear development. The Ly5.1 mouse strain appears to be the only rodent model so far identified with a high degree of the “human‐like” feature of unmyelinated SGNs that aggregate into neural clusters. Thus, this strain may provide a suitable animal platform for modeling human auditory information processing such as synchronous neural activity and other auditory response properties. J. Comp. Neurol. 518:3254–3271, 2010. © 2010 Wiley‐Liss, Inc.     

Substance P inhibits potassium and calcium currents in inner ear spiral ganglion neurons

Substance P (SP), a member of the tachykinin family of neurotransmitters and neuromodulators, has been identified on spiral ganglion neurons (SGNs) in the inner ear; however, its high affinity receptor, neurokinin-1 (NK1), has not been identified and the physiological effects of SP on SGNs are not well understood. To address these issues, immunolabeling, RT-PCR, Western blots and whole-cell patch-clamp recordings were made from SGNs in P0-P5 mouse cochlear organotypic cultures. The NK1 receptor was detected on SGNs by immunocytochemistry, the protein was detected in cochlear tissues by Western blots, and the mRNA for the NK1 receptor was also found in cochlear tissues of postnatal mice (P2) by RT-PCR. Application of SP (1 to 25 microM) significantly increased the latency of SGN action potentials (APs) (mean increase 7.8 +/- 4 ms; 25 microM of SP), prolonged the duration of the action potential and made the resting potential (RP) more positive (mean 9.0 +/- 7 mV) relative to normal values (-54 +/- 6 mV). SP (1 to 25 microM) also suppressed voltage-activated potassium currents (IK+) and calcium currents (ICa2+). Puffing 25 microM of SP onto SGNs suppressed IK+ by 43 +/- 9% (n = 7) and ICa2+ by 40.6 +/- 5.6% (n = 7); both currents recovered when SP was washed out. A SP antagonist blocked the SP-induced suppression of IK+ and ICa2+. These results indicate that SP acting through NK1 receptors can have direct neuromodulatory effects on SGNs.     

Interaction of neurotrophin signaling with Bcl‐2 localized to the mitochondria and endoplasmic reticulum on spiral ganglion neuron survival and neurite growth

Enhanced spiral ganglion neuron (SGN) survival and regeneration of peripheral axons following deafness will likely enhance the efficacy of cochlear implants. Overexpression of Bcl‐2 prevents SGN death but inhibits neurite growth. Here we assessed the consequences of Bcl‐2 targeted to either the mitochondria (GFP‐Bcl‐2‐Maob) or the endoplasmic reticulum (ER, GFP‐Bcl‐2‐Cb5) on cultured SGN survival and neurite growth. Transfection of wild‐type GFP‐Bcl‐2, GFP‐Bcl‐2‐Cb5, or GFP‐Bcl‐2‐Maob increased SGN survival, with GFP‐Bcl‐2‐Cb5 providing the most robust response. Paradoxically, expression of GFP‐Bcl‐2‐Maob results in SGN death in the presence of neurotrophin‐3 (NT‐3) and brain‐derived neurotrophic factor (BDNF), neurotrophins that independently promote SGN survival via Trk receptors. This loss of SGNs is associated with cleavage of caspase 3 and appears to be specific for neurotrophin signaling, insofar as coexpression of constitutively active mitogen‐activated kinase kinase (MEKΔEE) or phosphatidyl inositol‐3 kinase (P110), but not other prosurvival stimuli (e.g., membrane depolarization), also results in the loss of SGNs expressing GFP‐Bcl‐2‐Maob. MEKΔEE and P110 promote SGN survival, whereas P110 promotes neurite growth to a greater extent than NT‐3 or MEKΔEE. However, wild‐type GFP‐Bcl‐2, GFP‐Bcl‐2‐Cb5, and GFP‐Bcl‐2‐Maob inhibit neurite growth even in the presence of neurotrophins, MEKΔEE, or P110. Historically, Bcl‐2 has been thought to act primarily at the mitochondria to prevent neuronal apoptosis. Nevertheless, our data show that Bcl‐2 targeted to the ER is more effective at rescuing SGNs in the absence of trophic factors. Additionally, Bcl‐2 targeted to the mitochondria results in SGN death in the presence of neurotrophins. © 2010 Wiley‐Liss, Inc.     

Current concepts in cochlear ribbon synapse formation

In mammals, hair cells and spiral ganglion neurons (SGNs) in the cochlea together are sophisticated “sensorineural” structures that transduce auditory information from the outside world into the brain. Hair cells and SGNs are joined by glutamatergic ribbon‐type synapses composed of a molecular machinery rivaling in complexity the mechanoelectric transduction components found at the apical side of the hair cell. The cochlear hair cell ribbon synapse has received much attention lately because of recent and important findings related to its damage (sometimes termed “synaptopathy”) as a result of noise overexposure. During development, ribbon synapses between type I SGNs and inner hair cells form in the time window between birth and hearing onset and is a process coordinated with type I SGN myelination, spontaneous activity, synaptic pruning, and innervation by efferents. In this review, we highlight new findings regarding the diversity of type I SGNs and inner hair cell synapses, and the molecular mechanisms of selective hair cell targeting. Also discussed are cell adhesion molecules and protein constituents of the ribbon synapse, and how these factors participate in ribbon synapse formation. We also note interesting new insights into the morphological development of type II SGNs, and the potential for cochlear macrophages as important players in protecting SGNs. We also address recent studies demonstrating that the structural and physiological profiles of the type I SGNs do not reach full maturity until weeks after hearing onset, suggesting a protracted development that is likely modulated by activity.     

Increased proximal bifurcation of CA1 pyramidal apical dendrites in sema3A mutant mice

Semaphorin‐3A (Sema3A) is an attractive guidance molecule for cortical apical dendrites. To elucidate the role of Sema3A in hippocampal dendritic formation, we examined the Sema3A expression pattern in the perinatal hippocampal formation and analyzed hippocampal dendrites of the brains from young adult sema3A mutant mice. Sema3A protein was predominantly expressed in the hippocampal plate and the inner marginal zone at the initial period of apical dendritic growth. Neuropilin‐1 and plexin‐A, the receptor components for Sema3A, were also localized in the same regions. The Golgi impregnation method revealed that in wildtype mice more than 90% of hippocampal CA1 pyramidal neurons extended a single trunk or apical trunks bifurcated in stratum radiatum. Seven percent of the pyramidal neurons showed proximal bifurcation of apical trunks in stratum pyramidale or at the border of the stratum pyramidale and stratum radiatum. In sema3A mutant mice, proximally bifurcated apical dendrites were increased to 32%, while the single apical dendritic pyramidal neurons were decreased. We designate this phenotype in sema3A mutant mice as “proximal bifurcation.” In the dissociated culture system, approximately half of the hippocampal neurons from wildtype mice resembled pyramidal neurons, which possess a long, thick, and tapered dendrite. In contrast, only 30% of the neurons from sema3A mutants exhibited pyramidal‐like morphology. Proximal bifurcation of CA1 pyramidal neurons was also increased in the mutant mice of p35, an activator of cyclin‐dependent kinase 5 (Cdk5). Thus, Sema3A may facilitate the initial growth of CA1 apical dendrites via the activation of p35/Cdk5, which may in turn signal hippocampal development. J. Comp. Neurol. 516:360–375, 2009. © 2009 Wiley‐Liss, Inc.     

Prosurvival and proapoptotic intracellular signaling in rat spiral ganglion neurons in vivo after the loss of hair cells

Neurons depend on afferent input for survival. Rats were given daily kanamycin injections from P8 to P16 to destroy hair cells, the sole afferent input to spiral ganglion neurons (SGNs). Most SGNs die over an approximately 14-week period after deafferentation. During this period, the SGN population is heterogeneous. At any given time, some SGNs exhibit apoptotic markers--TUNEL and cytochrome c loss--whereas others appear nonapoptotic. We asked whether differences among SGNs in intracellular signaling relevant to apoptotic regulation could account for this heterogeneity. cAMP response element binding protein (CREB) phosphorylation, which reflects neurotrophic signaling, is reduced in many SGNs at P16, P23, and P32, when SGNs begin to die. In particular, nearly all apoptotic SGNs exhibit reduced phospho-CREB, implying that apoptosis is due to insufficient neurotrophic support. However, >32% of SGNs maintain high phospho-CREB levels, implying access to neurotrophic support. By P60, when approximately 50% of the SGNs have died, phospho-CREB levels in surviving neurons are not reduced, and SGN death is no longer correlated with reduced phospho-CREB. Activity in the proapoptotic Jun N-terminal kinase (JNK)-Jun signaling pathway is elevated in SGNs during the cell death period. This too is heterogeneous: <42% of the SGNs exhibited high phospho-Jun levels, but nearly all SGNs undergoing apoptosis exhibited elevated phospho-Jun. Thus, heterogeneity among SGNs in prosurvival and proapoptotic signaling is correlated with apoptosis. SGN death following deafferentation has an early phase in which apoptosis is correlated with reduced phospho-CREB and a later phase in which it is not. Proapoptotic JNK-Jun signaling is tightly correlated with SGN apoptosis.     

Membrane depolarization inhibits spiral ganglion neurite growth via activation of multiple types of voltage sensitive calcium channels and calpain

The effect of membrane electrical activity on spiral ganglion neuron (SGN) neurite growth remains unknown despite its relevance to cochlear implant technology. We demonstrate that membrane depolarization delays the initial formation and inhibits the subsequent extension of cultured SGN neurites. This inhibition depends directly on the level of depolarization with higher levels of depolarization causing retraction of existing neurites. Cultured SGNs express subunits for L-type, N-type, and P/Q type voltage-gated calcium channels (VGCCs) and removal of extracellular Ca(2+) or treatment with a combination of L-type, N-type, and P/Q-type VGCC antagonists rescues SGN neurite growth under depolarizing conditions. By measuring the fluorescence intensity of SGNs loaded with the fluorogenic calpain substrate t-butoxy carbonyl-Leu-Met-chloromethylaminocoumarin (20 microM), we demonstrate that depolarization activates calpains. Calpeptin (15 microM), a calpain inhibitor, prevents calpain activation by depolarization and rescues neurite growth in depolarized SGNs suggesting that calpain activation contributes to the inhibition of neurite growth by depolarization.     

Effects of Kir2.1 gene transfection in cochlear hair cells and application of neurotrophic factors on survival and neurite growth of co-cultured cochlear spiral ganglion neurons

Immature inner hair cells (IHCs) produce spontaneous action potentials, which may be associated with the survival of spiral ganglion neurons (SGNs) during early development. Later, this activity ceases in part by the expression of Kir channels. In the present study, SGNs were co-cultured with organ of Corti in which a Kir2.1 channel was over-expressed in an attempt to block the spontaneous activity of IHCs. The over-expression led to a reduced survival and neurite growth accompanied by increased SGN apoptosis. The enhanced activation of apoptosis was consistent with the inhibition of the survival-promoting pathway and the disruption of [Ca²⁺]_i homeostasis. Furthermore, the effect of Kir2.1 over-expression can be reversed by exogenous neurotrophic factors (NTFs). These results are consistent with the hypothesis that the earlier-than-normal expression of Kir2.1 in HCs inhibits their spontaneous activity required for SGN survival and neurite growth.     

Inner hair cells are not required for survival of spiral ganglion neurons in the adult cochlea

Studies of sensorineural hearing loss have long suggested that survival of spiral ganglion neurons (SGNs) depends on trophic support provided by their peripheral targets, the inner hair cells (IHCs): following ototoxic drugs or acoustic overexposure, IHC death is rapid whereas SGN degeneration is always delayed. However, recent noise-trauma studies show that SGNs can die even when hair cells survive, and transgenic mouse models show that supporting cell dysfunction can cause SGN degeneration in the absence of IHC pathology. To reexamine this issue, we studied a model of IHC loss that does not involve noise or ototoxic drugs. Mice lacking the gene for the high-affinity thiamine transporter (Slc19a2) have normal cochlear structure and function when fed a regular (thiamine-rich) diet. However, dietary thiamine restriction causes widespread, rapid (within 10 d) loss of IHCs. Using this model, we show that SGNs can survive for months after IHC loss, indicating that (1) IHCs are not necessary for neuronal survival, (2) neuronal loss in the other hearing loss models is likely due to effects of the trauma on the sensory neurons or other inner ear cells, and (3) that other cells, most likely supporting cells of the organ of Corti, are the main source of SGN survival factors. These results overturn a long-standing dogma in the study of sensorineural hearing loss and highlight the importance of cochlear supporting cells in neuronal survival in the adult inner ear.     

Progressive hearing loss in mice carrying a mutation in the p75 gene

The neurotrophin receptor p75 (p75NTR) is expressed in the developmental stage of the cochlea. However, the role of the p75NTR in the inner ear remains to be established. In this study, we conducted electrophysiological and morphological analyses of the auditory function of mice carrying a mutation in the p75 gene at different longitudinal stages. The mice carrying a mutation in the p75 gene showed an age-related progressive hearing loss. At 1 month, there was no obvious morphological change in the cochlea of the mice carrying a mutation in the p75 gene compared to wild-type mice, except for a slight loss of spiral ganglion neurons (SGNs). Auditory function was not significantly different between both genotypes from 1 to up to 4 months of age. The mice carrying a mutation in the p75 gene started to show progressive hearing loss at 4 months, when both SGN degeneration and hair cell (HC) loss were observed at the basal turn. These results suggest that the neurotrophin receptor p75 may play a significant role in the maintenance of cochlear function, and that mice carrying a mutation in the p75 gene could be a good animal model of early onset progressive hearing loss.     

Combining Cell-Based Therapies and Neural Prostheses to Promote Neural Survival

Cochlear implants provide partial restoration of hearing for profoundly deaf patients by electrically stimulating spiral ganglion neurons (SGNs); however, these neurons gradually degenerate following the onset of deafness. Although the exogenous application of neurotrophins (NTs) can prevent SGN loss, current techniques to administer NTs for long periods of time have limited clinical applicability. We have used encapsulated choroid plexus cells (NTCells; Living Cell Technologies, Auckland, New Zealand) to provide NTs in a clinically viable manner that can be combined with a cochlear implant. Neonatal cats were deafened and unilaterally implanted with NTCells and a cochlear implant. Animals received chronic electrical stimulation (ES) alone, NTs alone, or combined NTs and ES (ES + NT) for a period of as much as 8 months. The opposite ear served as a deafened unimplanted control. Chronic ES alone did not result in increased survival of SGNs or their peripheral processes. NT treatment alone resulted in greater SGN survival restricted to the upper basal cochlear region and an increased density of SGN peripheral processes. Importantly, chronic ES in combination with NTs provided significant SGN survival throughout a wider extent of the cochlea, in addition to an increased peripheral process density. Re-sprouting peripheral processes were observed in the scala media and scala tympani, raising the possibility of direct contact between peripheral processes and a cochlear implant electrode array. We conclude that cell-based therapy is clinically viable and effective in promoting SGN survival for extended durations of cochlear implant use. These findings have important implications for the safe delivery of therapeutic drugs to the cochlea.     

Opposing expression gradients of calcitonin‐related polypeptide alpha (Calca/Cgrpα) and tyrosine hydroxylase (Th) in type II afferent neurons of the mouse cochlea

Type II spiral ganglion neurons (SGNs) are small caliber, unmyelinated afferents that extend dendritic arbors hundreds of microns along the cochlear spiral, contacting many outer hair cells (OHCs). Despite these many contacts, type II afferents are insensitive to sound and only weakly depolarized by glutamate release from OHCs. Recent studies suggest that type II afferents may be cochlear nociceptors, and can be excited by ATP released during tissue damage, by analogy to somatic pain‐sensing C‐fibers. The present work compares the expression patterns among cochlear type II afferents of two genes found in C‐fibers: calcitonin‐related polypeptide alpha (Calca/Cgrpα), specific to pain‐sensing C‐fibers, and tyrosine hydroxylase (Th), specific to low‐threshold mechanoreceptive C‐fibers, which was shown previously to be a selective biomarker of type II versus type I cochlear afferents (Vyas et al., 2016 ). Whole‐mount cochlear preparations from 3‐week‐ to 2‐month‐old CGRPα‐EGFP (GENSAT) mice showed expression of Cgrpα in a subset of SGNs with type II‐like peripheral dendrites extending beneath OHCs. Double labeling with other molecular markers confirmed that the labeled SGNs were neither type I SGNs nor olivocochlear efferents. Cgrpα starts to express in type II SGNs before hearing onset, but the expression level declines in the adult. The expression patterns of Cgrpα and Th formed opposing gradients, with Th being preferentially expressed in apical and Cgrpα in basal type II afferent neurons, indicating heterogeneity among type II afferent neurons. The expression of Th and Cgrpα was not mutually exclusive and co‐expression could be observed, most abundantly in the middle cochlear turn.     

Molecular mechanisms underlying membrane-potential-mediated regulation of neuronal K2P2.1 channels

The activity of background K_2P channels adjusts the resting membrane potential to enable plasticity of excitable cells. Here we have studied the regulation of neuronal K_2P2.1 (KCNK2, TREK-1) channel activity by resting membrane potential. When heterologously expressed, K_2P2.1 currents gradually increased at hyperpolarizing potentials and declined at depolarizing potentials, with a midpoint potential of ? 60 mV. As K_2P channels are not equipped with an integral voltage sensor, we sought extrinsic cellular components that could convert changes in the membrane electrical field to cellular activity that would indirectly modify K_2P2.1 currents. We propose that membrane depolarization activated the Gq protein-coupled receptor pathway, in the apparent absence of ligand, resulting in phosphatidylinositol-4,5-bisphosphate (PIP₂) depletion through the action of phospholipase C. Our results suggest a novel mechanism in which an indirect pathway confers membrane potential regulation onto channels that are not intrinsically voltage sensitive to enhance regulation of neuronal excitability levels.     

Schwann cells revert to non-myelinating phenotypes in the deafened rat cochlea

Loss of sensory hair cells within the cochlea results in a permanent sensorineural hearing loss and initiates the gradual degeneration of spiral ganglion neurons (SGNs) - the primary afferent neurons of the cochlea. While these neurons are normally myelinated via Schwann cells, loss of myelin occurs as a precursor to neural degeneration. However, the relationship between demyelination and the status of Schwann cells in deafness is not well understood. We used a marker of peripheral myelin (myelin protein zero; P0) and a marker of Schwann cells (S100) to determine the temporal sequence of myelin and Schwann cell loss as a function of duration of deafness. Rat pups were systemically deafened for periods ranging from 2 weeks to greater than 6 months by co-administration of frusemide and gentamicin. Cochleae were cryosectioned and quantitative immunohistochemistry used to determine the extent of P0 and S100 labelling within the peripheral processes, SGN soma and their central processes within the modiolus. SGN density was also determined for each cochlear turn. P0 labelling decreased throughout the cochlea with increasing duration of deafness. The reduction in P0 labelling occurred at a faster rate than the SGN loss. In contrast, S100 labelling was not significantly reduced compared with age-matched controls in any cochlear region until 6 months post-deafening. These results suggest that Schwann cells may revert to non-myelinating phenotypes in response to deafness and exhibit greater survival traits than SGNs. The potential clinical significance of these findings for cochlear implants is discussed.     

Reduced excitability in the dentate gyrus network of βIV‐spectrin mutant mice in vivo

The submembrane cytoskeletal meshwork of the axon contains the scaffolding protein βIV‐spectrin. It provides mechanical support for the axon and anchors membrane proteins. Quivering (qv^3j) mice lack functional βIV‐spectrin and have reduced voltage‐gated sodium channel (VGSC) immunoreactivity at the axon initial segment and nodes of Ranvier. Because VGSCs are critically involved in action potential generation and conduction, we hypothesized that qv^3j mice should also show functional deficits at the network level. To test this hypothesis, we investigated granule cell function in the dentate gyrus of anesthetized qv^3j mice after electrical stimulation of the perforant path in vivo. This revealed an impaired input‐output relationship between stimulus intensity and granule cell population spikes and an enhanced paired‐pulse inhibition of population spikes, indicating a reduced ability of granule cells to generate action potentials and decreased network excitability. In contrast, the input‐output curve for evoked field excitatory postsynaptic potentials (fEPSPs) and paired‐pulse facilitation of fEPSPs were unchanged, suggesting normal excitatory synaptic transmission at perforant path‐granule cell synapses in qv^3j mutants. To corroborate our findings, we analyzed the influence of VGSC density reduction on dentate network activity using an established computational model of the dentate gyrus network. This in silico approach confirmed that the loss of VGSCs is sufficient to explain the electrophysiological changes observed in qv^3j mice. Taken together, our findings demonstrate that βIV‐spectrin is required for normal granule cell firing and for physiological levels of network excitability in the mouse dentate gyrus in vivo. © 2009 Wiley‐Liss, Inc.     

Electrophysiology of human muscle in culture

Human muscle obtained from biopsy specimens was grown in a primary tissue culture system and studied electrophysiologically. Myotubes with well-developed striations were formed after 3 to 5 weeks in culture. The myotubes had stable resting potentials (?50 mV), and generated all-or-none action potentials (92 mV amplitude) when stimulated. The resting permeability of the myotubes was found to be primarily dependent on K⁺ and to a lesser extent Na⁺ ($\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}\text{= 0.14 ± 0.06}$). Changes in external Cl^? did not affect the resting potential or resting conductance indicating that the Cl^? permeability is low compared with the permeabilities of K⁺ and Na⁺. A small but significant electrogenic NaK pump component to the resting potential was identified at low K⁺ concentrations (< 10 mm). The passive membrane parameters of myotubes were determined by analyzing the voltage decay transients of uniform fibers in response to the injection of a short hyperpolarizing pulse of current. The resting membrane resistance was 7780 ohm-cm²; membrane time constant, 22.9 ms; membrane capacitance, 3.8 μF; and electrotonic length, 1.1. The identification of the electrical characteristics of normal human muscle grown in primary tissue culture will serve as the basis for detailed comparative studies on diseased human muscle.     

The microbiome is essential for normal gut intrinsic primary afferent neuron excitability in the mouse

Background The role of intestinal microbiota in the development and function of host physiology is of high interest, especially with respect to the nervous system. While strong evidence has accrued that intestinal bacteria alter host nervous system function, mechanisms by which this occurs have remained elusive. For this reason, we have carried out experiments examining the electrophysiological properties of neurons in the myenteric plexus of the enteric nervous system (ENS) in germ‐free (GF) mice compared with specific pathogen‐free (SPF) control mice and adult germ‐free mice that have been conventionalized (CONV‐GF) with intestinal bacteria. Methods Segments of jejunum from 8 to 12 week old GF, SPF, and CONV‐GF mice were dissected to expose the myenteric plexus. Intracellular recordings in current‐clamp mode were made by impaling cells with sharp microelectrodes. Action potential (AP) shapes, firing thresholds, the number of APs fired at 2× threshold, and passive membrane characteristics were measured. Key Results In GF mice, excitability was decreased in myenteric afterhyperpolarization (AH) neurons as measured by a lower resting membrane potential and by the number of APs generated at 2× threshold. The post AP slow afterhyperpolarization (sAHP) was prolonged for GF compared with SPF and CONV‐GF animals. Passive membrane characteristics were also altered in GF mice by a decrease in input resistance. Conclusions & Inferences Here, we report the novel finding that commensal intestinal microbiota are necessary for normal excitability of gut sensory neurons and thus provide a potential mechanism for the transfer of information between the microbiota and nervous system.     

Properties of Single Calcium-activated Potassium Channels of Large Conductance in Rat Hippocampal Neurons in Culture

Patch-clamp recordings were made on rat hippocampal neurons maintained in culture. In cell-attached and excised inside-out and outside-out patches a large single-channel current was observed. This channel had a conductance of 220 and 100 pS in 140 mM [K⁺]_i/140 mM [K⁺]_o and 140 mM [K⁺]_i/3 mM [K⁺]_o respectively. From the reversal potential the channel was highly selective for K⁺, the P_K+/P_na+ ratio being 50/1. Channel activity was voltage-dependent, the open probability at 100 mM [Ca²⁺]_i increasing by e-fold for a 22 mV depolarization. It was also dependent on [Ca²⁺]_i at both resting and depolarized membrane potentials. Channel open states were best described by the sum of two exponentials with time constants that increased as the membrane potential became more positive. Channel activity was sensitive to both external (500 μM) and internal (5 mM) tetraethylammonium chloride. These data are consistent with the properties of maxi-K⁺ channels described in other preparations, and further suggest a role for maxi-channel activity in regulating neuronal excitability at the resting membrane potential. Channel activity was not altered by 8-chlorophenyl thio cAMP, concanavalin A, pH reduction or neuraminidase. In two of five patches lemakalim (BRL 38227) increased channel activity. Internal ruthenium red (10 μM) blocked the channel by shortening the duration of both open states. This change in channel gating was distinct from the ‘mode switching’ seen in two patches, where a channel switched spontaneously from normal activity typified by two open states to a mode where only short openings were represented.     

Periodic paralysis: Understanding channelopathies

Familial periodic paralyses are typical channelopathies (ie, caused by functional disturbances of ion channel proteins). The episodes of flaccid muscle weakness observed in these disorders are due to underexcitability of sarcolemma leading to a silent electromyogram and the lack of action potentials even upon electrical stimulation. Interictally, ion channel malfunction is well compensated, so that special exogenous or endogenous triggers are required to produce symptoms in the patients. An especially obvious trigger is the level of serum potassium (K⁺), the ion responsible for resting membrane potential and degree of excitability. The clinical symptoms can be caused by mutations in genes coding for ion channels that mediate different functions for maintaining the resting potential or propagating the action potential, the basis of excitability. The phenotype is determined by the type of functional defect brought about by the mutations, rather than the channel effected, because the contrary phenotypes hyperkalemic periodic paralysis (HyperPP) and hypokalemic periodic paralysis (HypoPP) may be caused by point mutations in the same gene. Still, the common mechanism for inexcitability in all known episodic-weakness phenotypes is a long-lasting depolarization that inactivates sodium ion (Na⁺) channels, initiating the action potential.