Voltage- and calcium-gated ion channels of neurons in the vertebrate retina

Highlights

• There are many types of voltage- and calcium-gated ion channels.

• There are almost 100 subtypes of retinal neurons that differ in their ion channels.

• Ion channel type and distribution shape responses of retinal neurons.

• Ion channel dysfunction can contribute to retinal disease.

Abstract

In this review, we summarize studies investigating the types and distribution of voltage- and calcium-gated ion channels in the different classes of retinal neurons: rods, cones, horizontal cells, bipolar cells, amacrine cells, interplexiform cells, and ganglion cells. We discuss differences among cell subtypes within these major cell classes, as well as differences among species, and consider how different ion channels shape the responses of different neurons. For example, even though second-order bipolar and horizontal cells do not typically generate fast sodium-dependent action potentials, many of these cells nevertheless possess fast sodium currents that can enhance their kinetic response capabilities. Ca²⁺ channel activity can also shape response kinetics as well as regulating synaptic release. The L-type Ca²⁺ channel subtype, Ca_V1.4, expressed in photoreceptor cells exhibits specific properties matching the particular needs of these cells such as limited inactivation which allows sustained channel activity and maintained synaptic release in darkness. The particular properties of K⁺ and Cl⁻ channels in different retinal neurons shape resting membrane potentials, response kinetics and spiking behavior. A remaining challenge is to characterize the specific distributions of ion channels in the more than 100 individual cell types that have been identified in the retina and to describe how these particular ion channels sculpt neuronal responses to assist in the processing of visual information by the retina.

Introduction

Investigators have explored the complement of ion channels in retinal neurons using an array of electrophysiological, immunohistochemical and molecular approaches. Early electrophysiological studies focused largely on non-mammalian vertebrates but later investigations provided greater insight into the properties of mammalian retinas. In recent years, the number of identified cell types in retina has increased considerably. For example, initial studies distinguished ON and OFF types of bipolar cells but we now recognize more than a dozen subtypes of bipolar cells. There is an even larger number of amacrine and ganglion cell types. Accompanying this expansion of recognized cell types has been a tremendous expansion in our understanding of the molecular diversity of ion channels. In that context, we thought it useful to summarize the current state of knowledge regarding the types of ion channels present in different types of retinal neurons. We focus on voltage- and Ca²⁺-dependent ion channels that transform photocurrents and synaptic currents into voltage responses. We do not focus on other ligand-gated ion channels such as the cyclic nucleotide-gated channels in photoreceptor outer segments or ion channels that couple directly to neurotransmitter receptors. Nor do we focus on aquaporins, gap junction hemichannels, TRP channels, or transporters. We use nomenclature recommended by the International Union of Pharmacology (IUPHAR) as summarized in “The Concise Guide to Pharmacology 2017/18”(Alexander et al., 2017a; Alexander et al., 2017b; Alexander et al., 2017c), supplemented by some of the more commonly used terms. Before turning to the different cell types, we begin with a summary of the subtypes and structural features of the ion channels that are the focus of this review.

K⁺ channels are formed from a tetrameric complex of 4 individual subunit proteins that each possess 2 transmembrane domains linked by a short pore-forming reentrant loop (P-loop) (Hibino et al., 2010; Tao et al., 2009). These channels lack a genuine voltage sensor but nevertheless exhibit an inwardly rectifying voltage-dependence that arises from blockade of outward currents by divalent cations at the intracellular surface of the channel pore. Some inwardly rectifying K⁺ channels (KIR1.1–7.1) are constitutively active, some are activated by Gβγ subunits of G-proteins (GIRK), and others are activated by a fall in intracellular ATP (K_ATP).

K⁺channels are formed from dimers with each subunit containing 4 transmembrane alpha helices (M1-4) along with two P-loops linking M1 to M2 and M3 to M4 (Brohawn et al., 2012; Miller and Long, 2012). The presence of two P-loops in each subunit endows this group with its name. Like KIR channels, two-pore channels (K2P1.1–12.1) lack a genuine voltage sensor. Constitutive activity of two pore channels contributes to the leak K⁺ current in many cells and is important for setting the resting membrane potential (Feliciangeli et al., 2015; Renigunta et al., 2015).

K⁺ channels (Armstrong, 2003; Kim and Nimigean, 2016; Kuang et al., 2015) are constructed from heteromeric or homomeric combinations of 4 individual subunits. Each subunit possesses 6 trans-membrane domains (S1–S6) with a P-loop located between S5 and S6. These channels are activated by depolarizing potentials. The voltage sensor in these and other similar voltage-dependent channels is the S4 trans-membrane domain that contains a number of positively charged amino acid residues (typically arginine). Membrane depolarization causes these residues to move towards the extracellular side of the membrane and the resulting conformational change in the protein opens the channel pore. It was originally proposed that voltage-sensing involves an outward helical screw motion of the S4 segment (Cha et al., 1999; Glauner et al., 1999), but subsequent structural analysis suggested that the S4 domain undergoes a paddle-like outward movement in response to depolarization (Jiang et al., 2003). Functional subtypes of voltage-gated K⁺ channels include delayed rectifier currents (I_KDR) in which outward currents inactivate slowly and A-type currents (I_KA) that inactivate rapidly. Rapid inactivation occurs through a “ball-and-chain” mechanism in which the amino terminus swings towards the channel pore to block conductance, involving either the K⁺ channel subunit itself or a segment of an accessory β subunit (Hille, 2001; Kurata and Fedida, 2006). Slow inactivation of I_KDR involves conformational changes that restrict pore conductance.

There are a few dozen subtypes of voltage-gated K⁺ channels (K_v1.1 to 12.3). K_v1-4 channels can form both homomeric and heteromeric channels with members of the same subclass (e.g., K_v1.1 with K_v1.2). Homomeric and heteromeric combinations of different K_v7 subunits form a special type of delayed rectifier current known as M-type currents. M currents were named for the ability of muscarinic agonists to inhibit these channels. Other agents that activate G_q/11 signaling pathways can also inhibit these channels (Brown and Passmore, 2009; Greene and Hoshi, 2017). K_v5, 6, 8 and 9 subunits have a similar structure as other K⁺ channels, but do not form functional homomeric channels. However, they can form functional channels in heteromeric combination with K_v2 subunits (Bocksteins, 2016).

K_v10-12 subunits encode ether-a-gogo (eag, K_v10), ether-a-gogo-related (erg, K_V11) and ether-a-gogo-like (elk, K_v12) channels (Bauer and Schwarz, 2018). Ether-a-go-go channels received their name because under ether anesthesia, Drosophilawith mutations in this channel shake their legs like go-go dancers (Vandenberg et al., 2012). These channels have a much shorter domain linking S4 and S5 domains compared to Kv1-2 channels that suggests a different gating mechanism (Whicher and MacKinnon, 2016). K_v10-12 channels have a C-terminal domain that is homologous to the cyclic nucleotide binding domain of CNG and HCN channels but lacks certain key residues so that it does not bind cyclic nucleotides.

In addition to the many pore-forming K_v channel subunits, a number of accessory K⁺ channel subunits have also been identified (Pongs and Schwarz, 2010). The many possible combinations of subunits and accessory proteins allows for an extremely large number of functionally and molecularly distinct K⁺ channels tuned to meet the particular needs of different cells.

K⁺ channels (Adelman et al., 2012; Christophersen and Wulff, 2015; Kaczmarek et al., 2017; Kshatri et al., 2018; Latorre et al., 2017) are functionally classified as small, intermediate and large conductance channels. Like voltage-gated K⁺ channels, Ca²⁺-activated K⁺ channels with small (K_Ca2.1–2.3; SK) and intermediate (K_Ca3.1; IK) single channel conductance are formed from four subunits, each containing 6 trans-membrane domains with one P-loop. Ca²⁺ activates these channels in a voltage-independent way by binding to calmodulin (CaM) associated with a CaM-binding domain on the C-terminus. Ca²⁺-activated K⁺ channels (K_Ca1.1) with a large single channel conductance (∼250 pS in symmetrical K⁺) are referred to as big K⁺ (BK) or Maxi K⁺ channels. In addition to the 6 transmembrane domains possessed by most other voltage-dependent channels, BK channels have an additional S0 trans-membrane domain, placing the N-terminus on the extracellular rather than the intracellular surface as is typical of channels with six transmembrane domains. In BK channels, binding of Ca²⁺ to domains on the intracellular surface can directly activate the channels (Yuan et al., 2011; Yuan et al., 2010). The accompanying allosteric changes to the protein also lower the threshold for voltage-dependent activation by shifting voltage-dependence to more negative potentials. There is only a single gene for BK channels, but as with other channels, there are multiple splice variants. Accessory beta and gamma subunits can further modify the activity of BK channels.

K⁺ channels (K_Na1.1–1.2) (Kaczmarek, 2013; Kaczmarek et al., 2017) are formed from 6 transmembrane domains and a P-loop, but the S4 segment appears less free to move and does not possess the sequence of positively charged amino acid characteristic of voltage-dependent K⁺ channels (Hite et al., 2015). Elevation of intracellular Na⁺ and Cl⁻ can both activate these channels. K_Na channels are expressed in many neurons but, to our knowledge, their presence in retinal neurons has not been investigated.

Voltage-gated Na⁺ (Na_V) channels are the key class of ion channels used to generate action potentials and are responsible for Na⁺ entry during the rising phase of the action potential (Ahern et al., 2016; Catterall, 2017). Unlike K⁺ channels that are formed from combinations of 2–4 individual subunits, the Na⁺ channel pore is formed from a single large α1 subunit protein. The α1 subunit consists of 4 similar sequences (I-IV), each possessing six transmembrane alpha helices (S1-6) with a short P-loop between S5 and S6, similar to individual voltage-dependent K⁺ channel subunits. As with most other voltage-dependent channels, the S4 domains function as the voltage sensor. Na⁺ channels underlying regenerative spiking are characterized by rapid and pronounced inactivation. Na⁺ channel inactivation involves a “hinged lid” mechanism in which the cytoplasmic loop between domains III and IV folds into the channel mouth to prevent conductance. There are currently 9 known isoforms of mammalian Na_V channel alpha subunits (Na_V1.1–1.9). Na_V1.1, Na_V1.2, and Na_V1.6 are highly expressed in neurons from the central nervous system including retinal ganglion cells. In addition to the α subunit, functional channels typically associate with β subunits that can modify voltage-sensitivity and gating of the channel.

Voltage-gated Ca²⁺ channels share a common structure with a large pore-forming α1 subunit that assembles with an intracellular β subunit and extracellular α2δ subunit (Catterall, 2011; Dolphin, 2016). Skeletal muscle channels (Ca_V1.1) also have accessory γ subunits but these do not appear to associate with Ca²⁺ channels in neurons. Similar to voltage-gated Na⁺ channels, the pore-forming α subunit is a single large protein composed of four domains each with six transmembrane alpha helices, a voltage sensor on the transmembrane segment S4 and a P-loop between S5 and S6. Ca²⁺ channels are functionally classified into two major classes: low- and high-voltage activated (LVA and HVA). LVA currents (Ca_V3.1–3.3) activate at more negative potentials than HVA currents. Because of their tiny single channel conductance and rapid inactivation resulting in transient currents, LVA currents are also referred to as T-type currents. HVA L-type currents (Ca_V1.1–1.4) were originally defined by their large single channel conductance and long-lasting activation due to slow inactivation. Pharmacologically, L-type Ca²⁺ currents (I_Ca) are selectively sensitive to dihydropyridine agonists (e.g., BayK8644) and antagonists (e.g., nifedipine). N-type currents (Ca_V2.2) are HVA channels that show intermediate properties between T and L-type channels. N-type currents were found to be neither too long-lasting nor too transient and N-type single channel conductance was neither too large nor too tiny. N-type currents are also predominantly expressed in neurons. Selective block of another current by funnel web spider toxin revealed additional HVA Ca²⁺ channels in cerebellar Purkinje cells (P-type). Keeping to this largely alphabetical arrangement, the next subtype identified by use of selective blockers was then named Q. P and Q type channels (Ca_V2.1) both derive from a single gene, CACNA1A. Finally, the residual current that remains after blocking the other HVA types with a cocktail of toxins was named R (Ca_V2.3).

HCN and CNG channels are cation channels that share considerable homology with other voltage-gated channels. The channels consist of 4 subunits that each possess 6 transmembrane domains (S1–S6) with a pore-forming P-loop between S5 and S6. The S4 segment contains a number of positively charged amino acids, but despite this similarity to other voltage-dependent channels, CNG channels show little or no voltage-dependence (James and Zagotta, 2018) and HCN channels (HCN1-4) are activated by membrane hyperpolarization rather than depolarization (Craven and Zagotta, 2006; Wahl-Schott and Biel, 2009). CNG and HCN channels have an intracellular cyclic nucleotide binding domain. CNG channels are opened by cyclic nucleotide binding and the voltage-dependence of HCN channels is strongly modulated by cyclic nucleotides (James and Zagotta, 2018).

HCN subunits form cation channels that are weakly selective for K⁺ over Na⁺ (P_Na/P_K = 0.2–0.3) and show little Ca²⁺ permeability. Unlike other voltage-gated ion channels, depolarization of HCN channels causes the S4 segment to move inward rather than outward towards the extracellular surface (Lee and MacKinnon, 2017). HCN channels are therefore activated by hyperpolarization and are typically active only at quite negative membrane potentials. Binding of cAMP can shift HCN voltage-dependence to more positive potentials and thereby promote HCN activity at membrane potentials that are more often attained under physiological conditions. HCN channel activity promotes oscillatory behavior in many neurons where it is sometimes referred to as an anomalous rectifier current (I_a). It also contributes to pacemaker currents in the heart where it is termed the “funny” current (I_f). In this review, we refer to the current carried by HCN channels as “I_h” for hyperpolarization-activated current.

Our focus is on voltage- and Ca²⁺-gated ion channels and so we touch only briefly on CNG channels. There are six mammalian subunits: CNGA1-3 form functional homotetrameric channels but CNGA4, CNGB1 and CNGB3 can only form functional channels in combination with CNGA1-3 subunits. CNG channels are non-selective for monovalent cations and also conduct Ca²⁺_, allowing it to serve as a second messenger in regulating phototransduction and olfactory transduction. We refer the interested reader to other reviews (Biel, 2009; Craven and Zagotta, 2006; James and Zagotta, 2018; Kaupp and Seifert, 2002).

Anoctamin 1 and 2 (Ano1 and 2, also known as TMEM16A and B) are Ca²⁺-activated Cl⁻ channels (Falzone et al., 2018; Kunzelmann, 2015; Whitlock and Hartzell, 2017). Ano1 and 2 are members of a larger family of anoctamin proteins (1–10) that also includes lipid scramblases and some cation channels. Ano1 and 2 anion channels are synergistically activated by voltage and Ca²⁺. The name “anoctamin” was given to TMEM proteins because it was originally thought that they possessed 8 transmembrane domains although it now appears that they have 10 transmembrane domains. Bestrophin proteins (Best1-4) can also form anion channels in expression system but there remains some question about whether these are truly Ca²⁺-activated Cl⁻ channels (Hartzell et al., 2008). Best1 is strongly expressed in retinal pigment epithelium cells and mutations in this protein can cause Best vitelliform macular dystrophy (Johnson et al., 2017).